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Sample records for peroxidase-labeled dna probes

  1. Strand displacement activated peroxidase activity of hemin for fluorescent DNA sensing.

    Science.gov (United States)

    Wang, Quanbo; Xu, Nan; Gui, Zhen; Lei, Jianping; Ju, Huangxian; Yan, Feng

    2015-10-07

    To efficiently regulate the catalytic activity of the peroxidase mimic hemin, this work designs a double-stranded DNA probe containing an intermolecular dimer of hemin, whose peroxidase activity can be activated by a DNA strand displacement reaction. The double-stranded probe is prepared by annealing two strands of hemin labelled DNA oligonucleotides. Using the fluorescent oxidation product of tyramine by H2O2 as a tracing molecule, the low peroxidase activity of the hemin dimer ensures a low fluorescence background. The strand displacement reaction of the target DNA dissociates the hemin dimer and thus significantly increases the catalytic activity of hemin to produce a large amount of dityramine for fluorescence signal readout. Based on the strand displacement regulated peroxidase activity, a simple and sensitive homogeneous fluorescent DNA sensing method is proposed. The detection can conveniently be carried out in a 96-well plate within 20 min with a detection limit of 0.18 nM. This method shows high specificity, which can effectively distinguish single-base mismatched DNA from perfectly matched target DNA. The DNA strand displacement regulated catalytic activity of hemin has promising application in the determination of various DNA analytes.

  2. Labelling of HBV-DNA probe using reagent made in China

    International Nuclear Information System (INIS)

    Wang Quanshi

    1991-01-01

    The labelling hepatitis Bvirus DNA (HBV-DNA) probe was studied by using reagent made in China. The results showed that: (1) The dNTPs with high specific activity was necessary for the labelling of nigh specific activity HBV-DNA probe; (2) reaction of labelling HBV-DNA probe was completed in a few minutes; (3) 0.37 MBq 3 H dTTP (specific activity 1.554TBq/mmol) was enough to label 1 μg HBV-DNA and the specific activity of probe reached 3.4 x 10 cpm/μg; (4) 7 MBqα- 32 P dATP (specific activity > 111 TBq/mmol) can label HBV-DNA probe to specific activity 1.35 x 10 cpm/μg. It was concluded that the reagent made in China can be used for the study in molecular biology

  3. Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

    International Nuclear Information System (INIS)

    Brakel, C.L.; Engelhardt, D.L.

    1985-01-01

    Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

  4. Leishmania diagnostic and identification py using 32P labelled DNA probes

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Melo, Maria Norma de

    1999-10-01

    P 32 labelled DNA probes are valious instruments for the parasitic diseases by using hybridization reaction. In this paper we describe the methodology and present the foundations for the radioactive probes production, based on the kinetoplast DNA (kDNA), for the Leishmania diagnostic an identification. We also describe the kDNA purification protocol from Leishmania reference cepa, the process of P 32 labelling of the kDNA by using the nick translation method, gathering, sample preparation and treatment, the optimum conditions for the hybridization reaction and the procedures for the autoradiography

  5. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

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    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  6. Detection of TTV-DNA in PBMC using digoxigenin labelled probe by in situ hybridization

    International Nuclear Information System (INIS)

    Liu Yang; Qi Qige

    2002-01-01

    To determine TTV-DNA in PBMC in patients with viral hepatitis, a study of in situ hybridization using digoxigenin labelled probe by PCR method to the TTV ORF1 region was performed on PBMC. Results showed that the detection rate of TTV-DNA using double-stranded probe in TTV-DNA positive group in sera was 58.06 (18/31), and the detection rate of TTV-DNA using double-stranded probe in TTV-DNA negative group in sera was 27.59 (8/29). For TTV-DNA positive group detected by double- stranded probe, then we use negative- stranded probe to detect their replication. The detection rate was 22.2%(4/18). Conclusions: TTV can infect PBMC and replicate in PBMC

  7. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  8. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  9. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    Science.gov (United States)

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

  10. Three-Dimensional Graphene Supported Bimetallic Nanocomposites with DNA Regulated-Flexibly Switchable Peroxidase-Like Activity.

    Science.gov (United States)

    Yuan, Fang; Zhao, Huimin; Zang, Hongmei; Ye, Fei; Quan, Xie

    2016-04-20

    A synergistic bimetallic enzyme mimetic catalyst, three-dimensional (3D) graphene/Fe3O4-AuNPs, was successfully fabricated which exhibited flexibly switchable peroxidase-like activity. Compared to the traditional 2D graphene-based monometallic composite, the introduced 3D structure, which was induced by the addition of glutamic acid, and bimetallic anchoring approach dramatically improved the catalytic activity, as well as the catalysis velocity and its affinity for substrate. Herein, Fe3O4NPs acted as supporters for AuNPs, which contributed to enhance the efficiency of electron transfer. On the basis of the measurement of Mott-Schottky plots of graphene and metal anchored hybrids, the catalysis mechanism was elucidated by the decrease of Fermi level resulted from the chemical doping behavior. Notably, the catalytic activity was able to be regulated by the adsorption and desorption of single-stranded DNA molecules, which laid a basis for its utilization in the construction of single-stranded DNA-based colorimetric biosensors. This strategy not only simplified the operation process including labeling, modification, and imprinting, but also protected the intrinsic affinity between the target and biological probe. Accordingly, based on the peroxidase-like activity and its controllability, our prepared nanohybrids was successfully adopted in the visualized and label-free sensing detections of glucose, sequence-specific DNA, mismatched nucleotides, and oxytetracycline.

  11. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  12. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  13. DNA probe labeling with digoxigenin-dUTP and its application in gene diagnosis

    International Nuclear Information System (INIS)

    Liu Guoyang

    1992-01-01

    DNA probe labeling by the randomly primed incorporation of digoxigenin-dUTP is reported. The sensitivity of color reaction and hybridization were 32 fg and 200 fg, respectively, and both were specific for the target. Single-copy and multi-copy gene fragments among 2 μg human genomic DNA were detected by β IVS II, Fr 3-42 and 3'HVR labeled with digoxigenin-dUTP. The results were consistent with a radioactive control assay. This method has been successfully used in the gene diagnosis of adult polycystic kidney disease

  14. DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Castelino, J

    1993-12-31

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with {sup 32}P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism`s genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens 10 figs, 2 tabs

  15. Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2008-02-01

    Full Text Available Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA.

  16. Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

    Science.gov (United States)

    He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

    2011-11-01

    The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

  17. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    Science.gov (United States)

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

  18. DNA stable-isotope probing (DNA-SIP).

    Science.gov (United States)

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  19. Horseradish peroxidase and antibody labeled gold nanoparticle probe for amplified immunoassay of ciguatoxin in fish samples based on capillary electrophoresis with electrochemical detection.

    Science.gov (United States)

    Zhang, Zhaoxiang; Liu, Ying; Zhang, Chaoying; Luan, Wenxiu

    2015-03-01

    This paper describes a new amplified immunoassay with horseradish peroxidase (HRP) and antibody (Ab) labeled gold nanoparticles (AuNPs) probe hyphenated to capillary electrophoresis (CE) with electrochemical (EC) detection for ultrasensitive determination of ciguatoxin CTX1B. AuNPs were conjugated with HRP and Ab, and then incubated with limited amount of CTX1B to produce immunocomplex. The immunoreactive sample was injected into capillary for CE separation and EC detection. Enhanced sensitivity was obtained by adopting the AuNPs as carriers of HRP and Ab at high HRP/Ab molar ratio. The calibration curve of CTX1B was in the range of 0.06-90 ng/mL. The detection limit was 0.045 ng/mL, which is 38-fold lower than that of HPLC-MS method for CTX1B analysis. The proposed method was successfully applied for the quantification of CTX1B in contamined fish samples by simultaneously labeling Ab and HRP on AuNPs. The amplified IA with HRP and Ab labeled AuNPs probe hyphenated to CE and EC detection provides a sensitive analytical approach for the determination of trace ciguatoxin in complex samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. DNA Probe for Lactobacillus delbrueckii

    Science.gov (United States)

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

  1. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

    International Nuclear Information System (INIS)

    Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

    1992-01-01

    Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

  2. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

    1992-01-01

    Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

  3. DNA probe for lactobacillus delbrueckii

    Energy Technology Data Exchange (ETDEWEB)

    Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  4. DNA probe for lactobacillus delbrueckii

    International Nuclear Information System (INIS)

    Delley, M.; Mollet, B.; Hottinger, H.

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α- 32 P-labeled probe

  5. Whole genomic DNA probe for detection of Porphyromonas endodontalis.

    Science.gov (United States)

    Nissan, R; Makkar, S R; Sela, M N; Stevens, R

    2000-04-01

    The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.

  6. The generation of radiolabeled DNA and RNA probes with polymerase chain reaction

    International Nuclear Information System (INIS)

    Schowalter, D.B.; Sommer, S.S.

    1989-01-01

    By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 X 10(9) cpm/micrograms or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence

  7. Sandwich nucleic acid hybridization: a method with a universally usable labeled probe for various specific tests

    International Nuclear Information System (INIS)

    Wolf, H.; Leser, U.; Haus, M.; Gu, S.Y.; Pathmanathan, R.

    1986-01-01

    The use of recombinant m13 phages as hybridization probes offers a considerable advantage over the commonly used recombinant plasmids as the preparation of the DNA probe is very simple and it can easily be labeled directly, e.g. with isotopes with long half-life like 125 I and used for hybridization. However, as the application of nucleic acid hybridization for diagnostic and epidemiological purposes becomes almost unavoidable, the logistic problems of keeping numerous individually labeled hybridization probes increase considerably and may reach prohibitory levels in less well-equipped laboratories. In a new sandwich technique, the first step involves hybridization with an unlabeled recombinant m13 DNA carrying an insert of the desired specificity. In a second step a universally usable labeled probe directed against the m13 part of the recombinant phage DNA is applied. This reduces considerably the problem of preparing and keeping multiple labeled probes in stock. (Auth.)

  8. A Fast, Sensitive and Label Free Electrochemical DNA Sensor

    International Nuclear Information System (INIS)

    Chen Yu; Elling; Lee Yokeling; Chong Serchoong

    2006-01-01

    A label free and sensitive DNA/RNA silicon based electrochemical microsensor array was developed by using thin film of the conducting polymer polypyrrole doped with an oligonucleotide probe. The electrochemical potential pulse amperometry technique was used for a biowarfare pathogen target DNA detection. The electrical potential assistanted DNA hybridisation method was applied. The sensor signal was increased by increasing the electrical potential assistanted DNA hybridisation time. It was possible to detect 0.34pmol and 0.072fmol of complementary oligonucleotide target in 0.1ml in seconds by using unpolished and polished gold electrode respectively. The probe preparation was also in seconds time, comparing indirect electrochemical DNA sensor, it has a fast sensor preparation as well as sensor response and label free advantages. The silicon microfabrication technique was used for this sensor array fabrication, which holds the potential to integrate with sensor electrical circuits. The conducting polymer polypyrrole was electrochemically deposited on each electrode respectively which has a possibility to dope the different DNA probe into the individual electrode to form a sensor array

  9. DNA imaging and quantification using chemi-luminescent probes

    International Nuclear Information System (INIS)

    Dorner, G.; Redjdal, N.; Laniece, P.; Siebert, R.; Tricoire, H.; Valentin, L.

    1999-01-01

    During this interdisciplinary study we have developed an ultra sensitive and reliable imaging system of DNA labelled by chemiluminescence. Based on a liquid nitrogen cooled CCD, the system achieves sensitivities down to 10 fg/mm 2 labelled DNA over a surface area of 25 x 25 cm 2 with a sub-millimeter resolution. Commercially available chemi-luminescent - and enhancer molecules are compared and their reaction conditions optimized for best signal-to-noise ratios. Double labelling was performed to verify quantification with radioactive probes. (authors)

  10. The cDNA sequence of a neutral horseradish peroxidase.

    Science.gov (United States)

    Bartonek-Roxå, E; Eriksson, H; Mattiasson, B

    1991-02-16

    A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

  11. Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

    Science.gov (United States)

    Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

    2014-07-15

    A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

  12. Development of DNA probes for Candida albicans

    International Nuclear Information System (INIS)

    Cheung, L.L.; Hudson, J.B.

    1988-01-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32 P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis

  13. Development of DNA probes for Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  14. Radioactively labelled DNA probes for crop improvement. Proceedings of a final research co-ordination meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-11-01

    With the advent of DNA molecular marker technology in the 1980s plant breeding had a new and powerful tool with which to increase its efficacy. Such markers are abundant and directly reveal information about the genotype and therefore are more useful than simple phenotypic markers. In plant breeding applications, molecular markers reveal information about variability and genetic relationships, and enable genetic mapping, which greatly assists the breeder in selection of parents and progeny, as well as in management of breeding strategies. Furthermore, molecular markers linked to phenotypic traits permit very early selection of superior progenies from breeding populations, therefore significantly reducing the need for field testing and greatly increasing efficiency of plant breeding programmes. For this to occur the oligonucleotide probes for labelling genetic markers and/or the primers for polymerase chain reactions to amplify genetic markers needed to be also accessible to scientists in developing Member States. In addition, technical information, training and troubleshooting were needed to support the utilization of DNA markers. In the early 1990s there was a dramatic increase in requests for access to this technology. This co-ordinated research project (CRP) facilitated the transfer of molecular marker technology, in terms of both material and information, from advanced laboratories to assist breeding programmes in developing countries. Two other CRPs were conducted concurrently in order to assist developing Member States to utilise molecular markers - Application of DNA Based Marker Mutations for Improvement of Cereals and other Sexually Reproduced Crop Plants, and Use of Novel DNA Fingerprinting Techniques for the Detection and Characterisation of Genetic Variation in Vegetatively Propagated Crops (IAEA-TECDOC-1010 and IAEA-TECDOC-1047, respectively). The present CRP built upon the success of the former projects by ensuring the availability of probes

  15. Radioactively labelled DNA probes for crop improvement. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    2001-11-01

    With the advent of DNA molecular marker technology in the 1980s plant breeding had a new and powerful tool with which to increase its efficacy. Such markers are abundant and directly reveal information about the genotype and therefore are more useful than simple phenotypic markers. In plant breeding applications, molecular markers reveal information about variability and genetic relationships, and enable genetic mapping, which greatly assists the breeder in selection of parents and progeny, as well as in management of breeding strategies. Furthermore, molecular markers linked to phenotypic traits permit very early selection of superior progenies from breeding populations, therefore significantly reducing the need for field testing and greatly increasing efficiency of plant breeding programmes. For this to occur the oligonucleotide probes for labelling genetic markers and/or the primers for polymerase chain reactions to amplify genetic markers needed to be also accessible to scientists in developing Member States. In addition, technical information, training and troubleshooting were needed to support the utilization of DNA markers. In the early 1990s there was a dramatic increase in requests for access to this technology. This co-ordinated research project (CRP) facilitated the transfer of molecular marker technology, in terms of both material and information, from advanced laboratories to assist breeding programmes in developing countries. Two other CRPs were conducted concurrently in order to assist developing Member States to utilise molecular markers - Application of DNA Based Marker Mutations for Improvement of Cereals and other Sexually Reproduced Crop Plants, and Use of Novel DNA Fingerprinting Techniques for the Detection and Characterisation of Genetic Variation in Vegetatively Propagated Crops (IAEA-TECDOC-1010 and IAEA-TECDOC-1047, respectively). The present CRP built upon the success of the former projects by ensuring the availability of probes

  16. Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

    Science.gov (United States)

    Wang, Xuewei; Qin, Wei

    2013-07-22

    The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

    International Nuclear Information System (INIS)

    Kimura, S.; Kotani, T.; McBride, O.W.; Umeki, K.; Hirai, K.; Nakayama, T.; Ohtaki, S.

    1987-01-01

    Two forms of human thyroid peroxidase cDNAs were isolated from a λgt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2

  18. Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

    Science.gov (United States)

    Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

    2016-05-01

    A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

  19. Impedimetric aptasensor for nuclear factor kappa B with peroxidase-like mimic coupled DNA nanoladders as enhancer.

    Science.gov (United States)

    Peng, Kanfu; Zhao, Hongwen; Xie, Pan; Hu, Shuang; Yuan, Yali; Yuan, Ruo; Wu, Xiongfei

    2016-07-15

    In this work, we developed a sensitive and universal aptasensor for nuclear factor kappa B (NF-κB) detection based on peroxidase-like mimic coupled DNA nanoladders for signal amplification. The dsDNA formed by capture DNA S1 and NF-κB binding aptamer (NBA) was firstly assembled on electrode surface. The presence of target NF-κB then led to the leave of NBA from electrode surface and thus provided the binding sites for immobilizing initiator to trigger in situ formation of DNA nanoladders on electrode surface. Since the peroxidase-like mimic manganese (III) meso-tetrakis (4-Nmethylpyridyl)-porphyrin (MnTMPyP) interacts with DNA nanoladders via groove binding, the insoluble benzo-4-chlorohexadienone (4-CD) precipitation derived from the oxidation of 4-chloro-1-naphthol (4-CN) could be formed on electrode surface in the presence of H2O2, resulting in a significantly amplified EIS signal output for quantitative target analysis. As a result, the developed aptasensor showed a low detection limit of 7pM and a wide linear range of 0.01-20nM. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective and stable detection of different types of target proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Technetium-99m labeled antisense probes uptake in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Zhang, Y.X.; Qin, G.M.; An, R.; Cao, G.X.; Cao, W.; Gao, Z.R.

    2002-01-01

    In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. The atherosclerotic plaques contained 3-4 fold more c-myc mRNA than those in the normal aortic arteries, while increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. Cell uptake studies: 99m Tc- MAG 3 -DNA radioactivity incorporation into porcine coronary smooth muscle cells in the log and plateau phases, respectively, was determined after different times of incubation at 37. The influence of extracellular 99m Tc- MAG 3 -DNA concentration on SMC uptake was also analyzed. [Results] Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The MAG 3 -DNA was labeled with 99m Tc at room temperature and neutral pH, with a mean labeling efficiency of 80.11%(s.d=2.96%,n=4). The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. After labeling, the stability of the DNA in saline or serum was retained as determined by reverse-phase Sep-Pak C18 chromatography analysis, except a shift at 30 min in serum incubation that suggesting a short time serum protein binding. 99m Tc-MAG 3 -c-myc uptake plateaued at 60 min and was directly proportional to the

  1. Imaging and high-sensitivity quantification of chemiluminescent labeled DNA-blots

    International Nuclear Information System (INIS)

    Dorner, G.

    1997-01-01

    The present thesis has for objective the development of both, methods of DNA labeling by chemiluminescence (via the catalytic activity of the enzyme alkaline phosphatase - AP) and an appropriate imaging system. Offering a competitive alternative to the detection of classical radio-labels in molecular-biological experiments of the blotting type, this technique should permit the realization of quantitative studies of gene expression at ultra-high sensitivity necessary in particular for differential-screening experiments. To reach our aim. we separated the project into three different parts. In a first step an imager based on a liquid-nitrogen-cooled CCD coupled to a standard optics (50 mm/fl.2) has been installed and characterized. This system offers a sensitive area of up to 625 cm 2 , a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm 2 labeled DNA. In a second part, the chemiluminescent light-generation process in solution has been investigated to optimize the parameters temperature. pH and concentration of the substrate as well as the enzyme. The substrate offering the highest light yield (CDP-Star in addition with the enhancer EMERALD II) allows quantification of AP down to 10 -15 M within a dynamic range of 10 4 in solution. Finally. preparation, immobilization and detection of AP-labeled DNA probes (via a biotin-streptavidin-biotin-AP bridge) on nylon membranes has been optimized. A linear relation between the light intensities and the amount of DNA was observed in a range of 10 fg/mm 2 - 100 pg/mm 2 . Hybridization of the probes to bacterial cloned target-DNA has been addressed after examination of the best hybridization conditions. Our protocol includes the treatment of a proteinase, which resulted in a significantly lower background on the filter. The results of our investigations suggest that the main conditions for a reliable differential-screening experiment are fulfilled when using

  2. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  3. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  4. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    International Nuclear Information System (INIS)

    Walia, S.; Khan, A.; Rosenthal, N.

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community

  5. A sensitive colorimetric aptasensor based on trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles.

    Science.gov (United States)

    Liu, Shuwen; Xu, Naihan; Tan, Chunyan; Fang, Wei; Tan, Ying; Jiang, Yuyang

    2018-08-14

    In this study, a novel colorimetric aptasensor was prepared by coupling trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles for highly sensitive and selective detection of target proteins. A three G-quadruplex (G4) DNA-hemin complex was employed as the trivalent peroxidase-mimic DNAzyme, in which hemin assisted the G4-DNA to fold into a catalytic conformation and act as an enzyme. The design of the aptasensor includes magnetic nanoparticles (MNPs), complementary DNA (cDNA) modified with biotin, and a label-free single strand DNA (ssDNA) including the aptamer and trivalent peroxidase-mimic DNAzyme. The trivalent DNAzyme, which has the highest catalytic activity among multivalent DNAzymes, catalyzed the H 2 O 2 -mediated oxidation of ABTS. The colorless ABTS was oxidized to produce a blue-green product that can be clearly distinguished by the naked eye. The aptamer and trivalent peroxidase-mimic DNAzyme promote the specificity and sensitivity of this detection method, which can be generalized for other targets by simply replacing the corresponding aptamers. To demonstrate the feasible use of the aptasensor for target detection, a well-known tumor biomarker MUC1 was evaluated as the model target. The limits of detection were determined to be 5.08 and 5.60 nM in a linear range of 50-1000 nM in a buffer solution and 10% serum system, respectively. This colorimetric and label-free aptasensor with excellent sensitivity and strong anti-interference ability has potential application in disease diagnoses, prognosis tracking, and therapeutic evaluation. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  7. "Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Kostečka, Pavel; Trefulka, Mojmír; Havran, Luděk; Paleček, Emil

    2007-01-01

    Roč. 79, č. 3 (2007), s. 1022-1029 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) IAA4004402; GA ČR(CZ) GA203/05/0043; GA ČR(CZ) GA203/04/1325; GA MPO(CZ) 1H-PK/42; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA labeling * osmium tetroxide complexes * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 5.287, year: 2007

  8. Labeled estrogens as mammary tumor probes

    International Nuclear Information System (INIS)

    Feenstra, A.

    1981-01-01

    In this thesis estrogens labeled with a gamma or positron emitting nuclide, called estrogen-receptor binding radiopharmaceuticals are investigated as mammary tumour probes. The requirements for estrogen-receptor binding radiopharmaceuticals are formulated and the literature on estrogens labeled for this purpose is reviewed. The potential of mercury-197/197m and of carbon-11 as label for estrogen-receptor binding radiopharmaceuticals is investigated. The synthesis of 197 Hg-labeled 4-mercury-estradiol and 2-mercury-estradiol and their properties in vitro and in vivo are described. It appears that though basically carbon-11 labeled compounds are very promising as mammary tumour probes, their achievable specific activity has to be increased. (Auth.)

  9. Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

    1994-09-01

    The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

  10. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

    2007-10-30

    We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

  11. Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

    International Nuclear Information System (INIS)

    Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L.

    1990-01-01

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32 P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 6 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains

  12. Absolute and direct microRNA quantification using DNA-gold nanoparticle probes.

    Science.gov (United States)

    Degliangeli, Federica; Kshirsagar, Prakash; Brunetti, Virgilio; Pompa, Pier Paolo; Fiammengo, Roberto

    2014-02-12

    DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

  13. Sequence- and structure-dependent DNA base dynamics: Synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

    International Nuclear Information System (INIS)

    Spaltenstein, A.; Robinson, B.H.; Hopkins, P.B.

    1989-01-01

    A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1 H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules

  14. DNA imaging and quantification using chemi-luminescent probes; Imagerie et quantification d`ADN par chimiluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Dorner, G; Redjdal, N; Laniece, P; Siebert, R; Tricoire, H; Valentin, L [Groupe I.P.B., Experimental Research Division, Inst. de Physique Nucleaire, Paris-11 Univ., 91 - Orsay (France)

    1999-11-01

    During this interdisciplinary study we have developed an ultra sensitive and reliable imaging system of DNA labelled by chemiluminescence. Based on a liquid nitrogen cooled CCD, the system achieves sensitivities down to 10 fg/mm{sup 2} labelled DNA over a surface area of 25 x 25 cm{sup 2} with a sub-millimeter resolution. Commercially available chemi-luminescent - and enhancer molecules are compared and their reaction conditions optimized for best signal-to-noise ratios. Double labelling was performed to verify quantification with radioactive probes. (authors) 1 fig.

  15. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely ...

  16. Multiplexed microRNA detection using lanthanide-labeled DNA probes and laser ablation inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    de Bang, Thomas Christian; Shah, Pratik; Cho, Seok Keun

    2014-01-01

    coupled plasma mass spectrometry (LA-ICPMS). Three miRNAs from Arabidopsis thaliana were analyzed simultaneously with high specificity, and the sensitivity of the method was comparable to radioactive detection (low femtomol range). The perspective of the developed method is highly multiplexed......In the past decade, microRNAs (miRNAs) have drawn increasing attention due to their role in regulation of gene expression. Especially, their potential as biomarkers in disease diagnostics has motivated miRNA research, including the development of simple, accurate, and sensitive detection methods....... The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively...

  17. Development of a biotinylated DNA probe for detection of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Deering, R.E.; Arakawa, C.K.; Oshima, K.H.; O'Hara, P.J.; Landolt, M.L.; Winton, J.R.

    1991-01-01

    A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \

  18. Polypyrrole–gold nanoparticle composites for highly sensitive DNA detection

    International Nuclear Information System (INIS)

    Spain, Elaine; Keyes, Tia E.; Forster, Robert J.

    2013-01-01

    DNA capture surfaces represent a powerful approach to developing highly sensitive sensors for identifying the cause of infection. Electrochemically deposited polypyrrole, PPy, films have been functionalized with electrodeposited gold nanoparticles to give a nanocomposite material, PPy–AuNP. Thiolated capture strand DNA, that is complementary to the sequence from the pathogen Staphylococcus aureus that causes mammary gland inflammation, was then immobilized onto the gold nanoparticles and any of the underlying gold electrode that is exposed. A probe strand, labelled with horse radish peroxidase, HRP, was then hybridized to the target. The concentration of the target was determined by measuring the current generated by reducing benzoquinone produced by the HRP label. Semi-log plots of the pathogen DNA concentration vs. faradaic current are linear from 150 pM to 1 μM and pM concentrations can be detected without the need for molecular, e.g., PCR or NASBA, amplification. The nanocomposite also exhibits excellent selectivity and single base mismatches in a 30 mer sequence can be detected

  19. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

    Science.gov (United States)

    Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

    2000-01-01

    A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

  20. Enzyme-enhanced fluorescence detection of DNA on etched optical fibers.

    Science.gov (United States)

    Niu, Shu-yan; Li, Quan-yi; Ren, Rui; Zhang, Shu-sheng

    2009-05-15

    A novel DNA biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylacetic acid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It is facile to regenerate this sensor through surface treatment with concentrated urea solution. It was discovered that the sensor can retain 70% of its original activity after three detection-regeneration cycles.

  1. Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

    Science.gov (United States)

    Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

    2014-06-15

    Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Macíčková-Cahová, Hana; Pivoňková, Hana; Špaček, Jan; Havran, Luděk; Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 9, č. 5 (2011), s. 1366-1371 ISSN 1477-0520 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk(CZ) LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : DNA tail- labelling * protein-DNA binding * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 3.696, year: 2011

  3. Double Antibody EIA of Cortisol Using Peroxidase As Label

    International Nuclear Information System (INIS)

    Karim, F.M.; Hamad, A.W.R.; Hashim, A.M.

    1998-01-01

    An enzyme immunoassay (EIA) technique for plasma cortisol was established by using cortisol-3 (carboxymethyl) oxime covalently linked to the horseradish peroxidase as the label. An antibody raised in the rabbits against cortisol-3-(carboxy-methyl) oxime-bovline serum albumin was used as the first anti-body. Sheep anti-rabbit gamma-globulin serum with 8 percent poly-ethyleneglycol were used to separate antibody-bound and free cortisol. The enzyme activity of the bound fraction was measured with ortho-phenylene diamine as substrate. The procedure performed at room temperature was evaluated by sensitivity (50 pg/ tube). The correlation coefficient between our enzyme immunoassay technique and radioimmunoassay technique for determination of plasma cortisol was 97 percent

  4. Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

    Science.gov (United States)

    Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

    2009-01-01

    A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

  5. Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

    Science.gov (United States)

    Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

    2014-09-01

    A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Biosensor for label-free DNA quantification based on functionalized LPGs.

    Science.gov (United States)

    Gonçalves, Helena M R; Moreira, Luis; Pereira, Leonor; Jorge, Pedro; Gouveia, Carlos; Martins-Lopes, Paula; Fernandes, José R A

    2016-10-15

    A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Multiply osmium-labeled reporter probes for electrochemical DNA hybridization assays: detection of trinucleotide repeats

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Havran, Luděk; Kizek, René; Paleček, Emil

    2004-01-01

    Roč. 20, č. 5 (2004), s. 985-994 ISSN 0956-5663 R&D Projects: GA MPO 1H-PK/42; GA AV ČR IAA4004108; GA AV ČR IBS5004355; GA AV ČR KJB4004302; GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z5004920 Keywords : electrochemical sensors * DNA hybridization * DNA labeling Subject RIV: BO - Biophysics Impact factor: 3.251, year: 2004

  8. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    International Nuclear Information System (INIS)

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  9. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

    Science.gov (United States)

    Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

    2001-01-01

    We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

  10. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  11. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    Science.gov (United States)

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  12. Use of avidin-biotin-peroxidase complex for measurement of UV lesions in human DNA by microELISA

    Energy Technology Data Exchange (ETDEWEB)

    Leipold, B [Technischen Universitaet Muenchen (Germany, F.R.). Dermatologische Klinik; Remy, W [Max-Planck-Institut fuer Biochemie, Muenchen (Germany, F.R.)

    1984-02-10

    The avidin/biotin system was introduced into the standard enzyme-linked immunosorbent assay (ELISA) to increase its sensitivity for detecting UV lesions in human DNA. Goat anti-rabbit IgG-peroxidase used in the standard ELISA as second antibody was replaced by biotinylated goat anti-rabbit IgG plus the avidin-biotin-peroxidase complex (ABC) reagent. Sensitivity of detection of plate-fixed UV-DNA-antibody complexes was increased about 8-fold and photolesions in human DNA samples irradiated with as low a dose as 1 J/m/sup 2/ UVC or a suberythermal dose of UVB light could be detected.

  13. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  14. Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

    Science.gov (United States)

    Reinhart, Wesley F.; Reifenberger, Jeff G.; Gupta, Damini; Muralidhar, Abhiram; Sheats, Julian; Cao, Han; Dorfman, Kevin D.

    2015-02-01

    We obtained experimental extension data for barcoded E. coli genomic DNA molecules confined in nanochannels from 40 nm to 51 nm in width. The resulting data set consists of 1 627 779 measurements of the distance between fluorescent probes on 25 407 individual molecules. The probability density for the extension between labels is negatively skewed, and the magnitude of the skewness is relatively insensitive to the distance between labels. The two Odijk theories for DNA confinement bracket the mean extension and its variance, consistent with the scaling arguments underlying the theories. We also find that a harmonic approximation to the free energy, obtained directly from the probability density for the distance between barcode labels, leads to substantial quantitative error in the variance of the extension data. These results suggest that a theory for DNA confinement in such channels must account for the anharmonic nature of the free energy as a function of chain extension.

  15. Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens

    International Nuclear Information System (INIS)

    Pickel, V.M.; Chan, J.; Milner, T.A.

    1986-01-01

    We examined whether autoradiographic localization of [ 125 I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [ 125 I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [ 125 I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH

  16. DNA-based stable isotope probing: a link between community structure and function

    International Nuclear Information System (INIS)

    Uhlik, Ondrej; Jecna, Katerina; Leigh, Mary Beth; Mackova, Martina; Macek, Tomas

    2009-01-01

    DNA-based molecular techniques permit the comprehensive determination of microbial diversity but generally do not reveal the relationship between the identity and the function of microorganisms. The first direct molecular technique to enable the linkage of phylogeny with function is DNA-based stable isotope probing (DNA-SIP). Applying this method first helped describe the utilization of simple compounds, such as methane, methanol or glucose and has since been used to detect microbial communities active in the utilization of a wide variety of compounds, including various xenobiotics. The principle of the method lies in providing 13C-labeled substrate to a microbial community and subsequent analyses of the 13C-DNA isolated from the community. Isopycnic centrifugation permits separating 13C-labeled DNA of organisms that utilized the substrate from 12C-DNA of the inactive majority. As the whole metagenome of active populations is isolated, its follow-up analysis provides successful taxonomic identification as well as the potential for functional gene analyses. Because of its power, DNA-SIP has become one of the leading techniques of microbial ecology research. But from other point of view, it is a labor-intensive method that requires careful attention to detail during each experimental step in order to avoid misinterpretation of results.

  17. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

    Science.gov (United States)

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

    2016-08-16

    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

  18. Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

    Energy Technology Data Exchange (ETDEWEB)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan (Malaysia); Ahmad, Haslina; Harun, Siti Norain [Chemistry Department, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor (Malaysia)

    2014-09-03

    A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  19. A polymeric liquid membrane electrode responsive to 3,3',5,5'-tetramethylbenzidine oxidation for sensitive peroxidase/peroxidase mimetic-based potentiometric biosensing.

    Science.gov (United States)

    Wang, Xuewei; Yang, Yangang; Li, Long; Sun, Mingshuang; Yin, Haogen; Qin, Wei

    2014-05-06

    The oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) has great utility in bioanalysis such as peroxidase/peroxidase mimetic-based biosensing. In this paper, the behaviors of TMB oxidation intermediates/products in liquid/liquid biphasic systems have been investigated for the first time. The free radical, charge transfer complex, and diimine species generated by TMB oxidation are all positively charged under acidic and near-neutral conditions. Electron paramagnetic resonance and visible absorbance spectroscopy data demonstrate that these cationic species can be effectively transferred from an aqueous phase into a water-immiscible liquid phase functionalized by an appropriate cation exchanger. Accordingly, sensitive potential responses of TMB oxidation have been obtained on a cation exchanger-doped polymeric liquid membrane electrode under mildly acidic and near-neutral conditions. By using the membrane electrode responsive to TMB oxidations, two sensitive potentiometric biosensing schemes including the peroxidase-labeled sandwich immunoassay and G-quadruplex DNAzyme-based DNA hybridization assay have been developed. The obtained detection limits for the target antigen and DNA are 0.02 ng/mL and 0.1 nM, respectively. Coupled with other advantages such as low cost, high reliability, and ease of miniaturization and integration, the proposed polymeric liquid membrane electrode holds great promise as a facile and efficient transducer for TMB oxidation and related biosensing applications.

  20. Electrochemical detection of human papillomavirus DNA type 16 using a pyrrolidinyl peptide nucleic acid probe immobilized on screen-printed carbon electrodes.

    Science.gov (United States)

    Jampasa, Sakda; Wonsawat, Wanida; Rodthongkum, Nadnudda; Siangproh, Weena; Yanatatsaneejit, Pattamawadee; Vilaivan, Tirayut; Chailapakul, Orawon

    2014-04-15

    An electrochemical biosensor based on an immobilized anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe was successfully developed for the selective detection of human papillomavirus (HPV) type 16 DNA. A 14-mer acpcPNA capture probe was designed to recognize a specific 14 nucleotide region of HPV type 16 L1 gene. The redox-active label anthraquinone (AQ) was covalently attached to the N-terminus of the acpcPNA probe through an amide bond. The probe was immobilized onto a chitosan-modified disposable screen-printed carbon electrode via a C-terminal lysine residue using glutaraldehyde as a cross-linking agent. Hybridization with the target DNA was studied by measuring the electrochemical signal response of the AQ label using square-wave voltammetric analysis. The calibration curve exhibited a linear range between 0.02 and 12.0 µM with a limit of detection and limit of quantitation of 4 and 14 nM, respectively. This DNA sensing platform was successfully applied to detect the HPV type 16 DNA from a PCR amplified (240 bp fragment of the L1 gene) sample derived from the HPV type 16 positive human cancer cell line (SiHa), and failed to detect the HPV-negative c33a cell line. The sensor probe exhibited very high selectivity for the complementary 14 base oligonucleotide over the non-complementary oligonucleotides with sequences derived from HPV types 18, 31 and 33. The proposed sensor provides an inexpensive tool for the early stage detection of HPV type 16, which is an important biomarker for cervical cancer. © 2013 Elsevier B.V. All rights reserved.

  1. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhong Feng; Chen, Dong Mei [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2015-10-15

    Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. - Highlights: • Conformational switch of i-motif is used for the detection of glucose and urea. • The sensor can be regenerated. • The proposed method is successfully applied in real sample assay. • Our method is label-free and inexpensive.

  2. Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

    International Nuclear Information System (INIS)

    Lin, H.J.; Chung, H.T.; Lai, C.L.; Leong, S.; Tam, O.S.

    1989-01-01

    A novel assay for supercoiled and other fully double-stranded forms of hepatitis B virus (HBV) DNA in blood is presented that utilizes molecular hybridisation to a radiophosphorous-labeled oligonucleotide probe. The probe [5'-d(ACGTGCAGAGGTGAAGCGA)] is complementary to the S(+)-strand sequence furthest downstream, at the end of the gap. We examined blood specimens from 137 healthy HBsAg-positive individuals, applying the probe to dots representing 2-3.5 ml serum or plasma. We found that supercoiled HBV is present in many HBV DNA-positive blood specimens albeit in small quantities. Of the 104 specimens that were positive for HBV DNA of any form, 53 annealed to the probe. Serial specimens from the same subject taken over a period of months showed that the proportion of supercoil to other HBV DNA forms was variable. The presence of supercoil HBV DNA was not closely correlated with the level of serum HBV DNA polymerase. The supercoil is an HBV DNA form that can persist in the liver in the presence or absence of other replicative intermediates. This assay may enable further characterization of the status of HBV infection

  3. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    黄承志; 李原芳; 黄新华; 范美坤

    2000-01-01

    The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  4. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The microarray of DNA probes with 5′-NH2 and 5′-Tex/3′-NH2 modified terminus on 10 m m carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is characterized in the present paper. It was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentration of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  5. [Application of DNA labeling technology in forensic botany].

    Science.gov (United States)

    Znang, Xian; Li, Jing-Lin; Zhang, Xiang-Yu

    2008-12-01

    Forensic botany is a study of judicial plant evidence. Recently, researches on DNA labeling technology have been a mainstream of forensic botany. The article systematically reviews various types of DNA labeling techniques in forensic botany with enumerated practical cases, as well as the potential forensic application of each individual technique. The advantages of the DNA labeling technology over traditional morphological taxonomic methods are also summarized.

  6. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    Science.gov (United States)

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  7. Gene probes: principles and protocols

    National Research Council Canada - National Science Library

    Aquino de Muro, Marilena; Rapley, Ralph

    2002-01-01

    ... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

  8. A specific DNA probe which identifies Babesia bovis in whole blood.

    Science.gov (United States)

    Petchpoo, W; Tan-ariya, P; Boonsaeng, V; Brockelman, C R; Wilairat, P; Panyim, S

    1992-05-01

    A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

  9. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen (Germany); Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)

    2016-07-01

    Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

  10. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

    International Nuclear Information System (INIS)

    Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

    2016-01-01

    Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

  11. Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

    International Nuclear Information System (INIS)

    Zaffino, R L; Mir, M; Samitier, J

    2014-01-01

    We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications. (paper)

  12. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J

    1992-01-01

    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...

  13. Graphene-palladium nanowires based electrochemical sensor using ZnFe2O4-graphene quantum dots as an effective peroxidase mimic.

    Science.gov (United States)

    Liu, Weiyan; Yang, Hongmei; Ma, Chao; Ding, Ya-nan; Ge, Shenguang; Yu, Jinghua; Yan, Mei

    2014-12-10

    We proposed an electrochemical DNA sensor by using peroxidase-like magnetic ZnFe2O4-graphene quantum dots (ZnFe2O4/GQDs) nanohybrid as a mimic enzymatic label. Aminated graphene and Pd nanowires were successively modified on glassy carbon electrode, which improved the electronic transfer rate as well as increased the amount of immobilized capture ssDNA (S1). The nanohybrid ZnFe2O4/GQDs was prepared by assembling the GQDs on the surface of ZnFe2O4 through a photo-Fenton reaction, which was not only used as a mimic enzyme but also as a carrier to label complementary ssDNA (S3). By synergistically integrating highly catalytically activity of nano-sized GQDs and ZnFe2O4, the nanohybrid possessed highly-efficient peroxidase-like catalytic activity which could produce a large current toward the reduction of H2O2 for signal amplification. Thionine was used as an excellent electron mediator. Compared with traditional enzyme labels, the mimic enzyme ZnFe2O4/GQDs exhibited many advantages such as environment friendly and better stability. Under the optimal conditions, the approach provided a wide linear range from 10(-16) to 5×10(-9) M and low detection limit of 6.2×10(-17) M. The remarkable high catalytic capability could allow the nanohybrid to replace conventional peroxidase-based assay systems. The new, robust and convenient assay systems can be widely utilized for the identification of other target molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Probe Selection for DNA Microarrays using OligoWiz

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Juncker, Agnieszka; Nielsen, Henrik Bjørn

    2007-01-01

    Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client-server appl......Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client......-server application that offers a detailed graphical interface and real-time user interaction on the client side, and massive computer power and a large collection of species databases (400, summer 2007) on the server side. Probes are selected according to five weighted scores: cross-hybridization, deltaT(m), folding...... computer skills and can be executed from any Internet-connected computer. The probe selection procedure for a standard microarray design targeting all yeast transcripts can be completed in 1 h....

  15. Graphene–palladium nanowires based electrochemical sensor using ZnFe2O4–graphene quantum dots as an effective peroxidase mimic

    International Nuclear Information System (INIS)

    Liu, Weiyan; Yang, Hongmei; Ma, Chao; Ding, Ya-nan; Ge, Shenguang; Yu, Jinghua; Yan, Mei

    2014-01-01

    Highlights: • The nanohybrid ZnFe 2 O 4 /GQDs was developed by assembling the GQDs on the ZnFe 2 O 4 through a photo-Fenton reaction. • The ZnFe 2 O 4 /GQDs exhibited higher peroxidase-like activity and better stability than each individual and HRP. • An electrochemical sensor was fabricated using ZnFe 2 O 4 /GQDs nanohybrid as a mimic enzymatic to detect DNA. • Graphene and Pd nanowires were modified on the glassy carbon electrode, which improved the electronic transfer rate. - Abstract: We proposed an electrochemical DNA sensor by using peroxidase-like magnetic ZnFe 2 O 4 –graphene quantum dots (ZnFe 2 O 4 /GQDs) nanohybrid as a mimic enzymatic label. Aminated graphene and Pd nanowires were successively modified on glassy carbon electrode, which improved the electronic transfer rate as well as increased the amount of immobilized capture ssDNA (S1). The nanohybrid ZnFe 2 O 4 /GQDs was prepared by assembling the GQDs on the surface of ZnFe 2 O 4 through a photo-Fenton reaction, which was not only used as a mimic enzyme but also as a carrier to label complementary ssDNA (S3). By synergistically integrating highly catalytically activity of nano-sized GQDs and ZnFe 2 O 4 , the nanohybrid possessed highly-efficient peroxidase-like catalytic activity which could produce a large current toward the reduction of H 2 O 2 for signal amplification. Thionine was used as an excellent electron mediator. Compared with traditional enzyme labels, the mimic enzyme ZnFe 2 O 4 /GQDs exhibited many advantages such as environment friendly and better stability. Under the optimal conditions, the approach provided a wide linear range from 10 −16 to 5 × 10 −9 M and low detection limit of 6.2 × 10 −17 M. The remarkable high catalytic capability could allow the nanohybrid to replace conventional peroxidase-based assay systems. The new, robust and convenient assay systems can be widely utilized for the identification of other target molecules

  16. Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

    Directory of Open Access Journals (Sweden)

    Cheng Zhang

    2017-04-01

    Full Text Available Quantum dots (QDs are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.

  17. Rapid on-site detection of Acidovorax avenae subsp. citrulli by gold-labeled DNA strip sensor.

    Science.gov (United States)

    Zhao, Wenjun; Lu, Jie; Ma, Wenwei; Xu, Chuanlai; Kuang, Hua; Zhu, Shuifang

    2011-06-15

    Acidovorax avenae subsp. citrulli (AAC) is one of the most harmful diseases in cucurbit production. A rapid and sensitive DNA strip sensor was constructed based on gold nanoparticle-labeled oligonucleotide probes for the detection of AAC. Both the qualitative and semi-quantitative detections of target DNA were successfully achieved using the developed DNA strip sensor. The qualitative limit of detection (LOD) of the strip sensor was determined as 4 nM. The LOD for the semi-quantitative detection was calculated to be 0.48 nM in the range of 0-10 nM. The genomic DNA was detected directly using the DNA strip sensor without any further treatment. This DNA strip sensor is a potentially useful tool for rapid on-site DNA screening. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Hg(2+) detection using a phosphorothioate RNA probe adsorbed on graphene oxide and a comparison with thymine-rich DNA.

    Science.gov (United States)

    Huang, Po-Jung Jimmy; van Ballegooie, Courtney; Liu, Juewen

    2016-06-07

    Mercury is a highly toxic heavy metal and many DNA-based biosensors have been recently developed for Hg(2+) detection in water. Among them, thymine-rich DNA is the most commonly used for designing Hg(2+) sensors. However, the thymine-Hg(2+) interaction is strongly affected by the buffer conditions. We recently reported a molecular beacon containing phosphorothioate (PS)-modified RNA linkages that can be cleaved by Hg(2+). In this work, the fluorescence quenching and DNA adsorption properties of nano-sized graphene oxide (NGO) were used to develop a new sensor using the PS-RNA chemistry. Three DNA probes, containing one, three and five PS-RNA linkages, respectively, were tested. Finally, a fluorophore-labeled poly-A DNA with five PS-RNA linkages was selected and adsorbed by NGO. In the presence of Hg(2+), the fluorophore was released from NGO due to the cleavage reaction, resulting in a fluorescence enhancement. This sensor is highly selective for Hg(2+) with a detection limit of 8.5 nM Hg(2+). For comparison, a fluorophore-labeled poly-T DNA was also tested, which responded to Hg(2+) more slowly and was inhibited by high NaCl concentrations, while the PS-RNA probe was more tolerant to different buffer conditions. This work indicates a new method for interfacing DNA with NGO for Hg(2+) detection.

  19. All-atom molecular dynamics simulations of spin labelled double and single-strand DNA for EPR studies.

    Science.gov (United States)

    Prior, C; Danilāne, L; Oganesyan, V S

    2018-05-16

    We report the first application of fully atomistic molecular dynamics (MD) simulations to the prediction of electron paramagnetic resonance (EPR) spectra of spin labelled DNA. Models for two structurally different DNA spin probes with either the rigid or flexible position of the nitroxide group in the base pair, employed in experimental studies previously, have been developed. By the application of the combined MD-EPR simulation methodology we aimed at the following. Firstly, to provide a test bed against a sensitive spectroscopic technique for the recently developed improved version of the parmbsc1 force field for MD modelling of DNA. The predicted EPR spectra show good agreement with the experimental ones available from the literature, thus confirming the accuracy of the currently employed DNA force fields. Secondly, to provide a quantitative interpretation of the motional contributions into the dynamics of spin probes in both duplex and single-strand DNA fragments and to analyse their perturbing effects on the local DNA structure. Finally, a combination of MD and EPR allowed us to test the validity of the application of the Model-Free (M-F) approach coupled with the partial averaging of magnetic tensors to the simulation of EPR spectra of DNA systems by comparing the resultant EPR spectra with those simulated directly from MD trajectories. The advantage of the M-F based EPR simulation approach over the direct propagation techniques is that it requires motional and order parameters that can be calculated from shorter MD trajectories. The reported MD-EPR methodology is transferable to the prediction and interpretation of EPR spectra of higher order DNA structures with novel types of spin labels.

  20. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Moreno, Elizabeth Castro; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela; Fernandes, Octavio

    2002-01-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with 32 P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with 32 P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  1. Reverse sample genome probing, a new technique for identification of bacteria in environmental samples by DNA hybridization, and its application to the identification of sulfate-reducing bacteria in oil field samples

    International Nuclear Information System (INIS)

    Voordouw, G.; Voordouw, J.K.; Karkhoff-Schweizer, R.R.; Fedorak, P.M.; Westlake, D.W.S.

    1991-01-01

    A novel method for identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a standard) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples

  2. High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

    Science.gov (United States)

    Weier, Heinz-Ulli G.; Munne, S.; Lersch, Robert A.; Marquez, C.; Wu, J.; Pedersen, Roger A.; Fung, Jingly

    1999-06-01

    The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in

  3. Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

    Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

  4. Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

    Science.gov (United States)

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2007-06-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

  5. Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

    Science.gov (United States)

    Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

    1991-02-15

    The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

  6. Branched-DNA signal amplification combined with paper chromatography hybridization assay and used in hepatitis B virus DNA detection

    International Nuclear Information System (INIS)

    Fu, F.Z.; Liu, L.X.; Wang, W.Q.; Sun, S. H.; Liu, L.B.

    2002-01-01

    Nucleic acids detection method is vital to the clinical pathogen diagnosis. The established method can be classified into target direct amplification and signal amplification format according to the target DNA or RNA being directly amplified or not. Those methods have advantages and disadvantages respectively in the clinical application. In the United States of American, branched-DNA as a strong signal amplifier is broadly used in the quantification of the nucleic acids. To gain satisfied sensitivity, some expensive label molecular and instruments should be adopted. Personnel should be special trained to perform. Hence, those can't be widely carried out in the Third World. To avoid those disadvantages, we used the branched-DNA amplifier in the paper chromatography hybridization assay. Methods: Branched DNA signal amplifier and series of probes complementary to the nucleic acid sequence of hepatitis B virus (HBV) have been synthesized. HBV-DNA or it's capture probe were immobilized on the high flow nitrocellulose strip. Having loaded at one end of the strip in turn, probes or HBV-DNA in the hybridization solution migrate to the opposite end of the strip by capillary forces and hybridizes to the immobilized DNA. The branched-DNA signal amplifier and probe labeled with biotin or 32P were then loaded. Through streptavidin-alkaline phosphatase (SA-AP) conjugate and NBT/BCIP ( the specific chromogenic substrate of AP) or autoradiography, the result can be visualized by color reaction or image production on the X-ray film. Results: The sensitivity of this HBV-DNA detection method used probe labeled with biotin and 32P are 1ng and 10pg. The method using the probe labeled with biotin is simple and rapid (2h) without depending on special instruments, it also avoids the pollution of EtBr which can lead to tumor. And the method using the probe labeled with 32P is simple and sensitive, with the exception of long time autoradiography and the inconvenient isotopic disposal

  7. Genotypic characterization of Rickettsiae by DNA probes generated from Rickettsia Prowazekii DNA

    International Nuclear Information System (INIS)

    Demkin, V.V.; Rydkina, E.B.; Likhoded, L.Ya.; Ignatovich, V.F.; Genig, V.A.; Balayeva, N.M.

    1994-01-01

    Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species were of the spotted fever group (SFG)rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragment of R prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsudamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization pattern with TG species and R. akari. PBH11. PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intra-species pattern were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification. (author)

  8. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

    Science.gov (United States)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  9. Multiple tag labeling method for DNA sequencing

    Science.gov (United States)

    Mathies, R.A.; Huang, X.C.; Quesada, M.A.

    1995-07-25

    A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

  10. Development of a sensitive electrochemical DNA sensor by 4-aminothiophenol self-assembled on electrodeposited nanogold electrode coupled with Au nanoparticles labeled reporter ssDNA

    International Nuclear Information System (INIS)

    Li Guangjiu; Liu Lihua; Qi Xiaowei; Guo Yaqing; Sun Wei; Li Xiaolin

    2012-01-01

    Graphical abstract: - Abstract: A novel and sensitive electrochemical DNA biosensor was fabricated by using the 4-aminothiophenol (4-ATP) self-assembled on electrodeposited gold nanoparticles (NG) modified electrode to anchor capture ssDNA sequences and Au nanoparticles (AuNPs) labeled with reporter ssDNA sequences, which were further coupled with electroactive indicator of hexaammineruthenium (III) ([Ru(NH 3 ) 6 ] 3+ ) to amplify the electrochemical signal of hybridization reaction. Different modified electrodes were prepared and characterized by cyclic voltammetry, scanning electron microscope and electrochemical impedance spectroscopy. By using a sandwich model for the capture of target ssDNA sequences, which was based on the shorter probe ssDNA and AuNPs label reporter ssDNA hybridized with longer target ssDNA, the electrochemical behavior of [Ru(NH 3 ) 6 ] 3+ was monitored by differential pulse voltammetry (DPV). The fabricated electrochemical DNA sensor exhibited good distinguish capacity for the complementary ssDNA sequence and two bases mismatched ssDNA. The dynamic detection range of the target ssDNA sequences was from 1.4 × 10 −11 to 2.0 × 10 −9 mol/L with the detection limit as 9.5 × 10 −12 mol/L (3σ). So in this paper a new electrochemical DNA sensor was designed with gold nanoparticles as the immobilization platform and the signal amplifier simultaneously.

  11. Electrochemical gene sensor for Mycoplasma pneumoniae DNA using dual signal amplification via a Pt-Pd nanowire and horse radish peroxidase

    International Nuclear Information System (INIS)

    Liu, Linlin; Xiang, Guiming; Jiang, Dongneng; Du, Chunlan; Liu, Chang; Huang, Weiwei; Pu, Xiaoyun

    2016-01-01

    A dually amplified DNA biosensor was constructed for the determination of the DNA of Mycoplasma pneumoniae (M. pneu). A gold electrode was modified with 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA; a π-stacking perylene semiconductor dye with outstanding electronic and optical properties), a layer of gold nanoparticles (nano-Au), and capture DNA. Pt-Pd nanowires served as carriers for the co immobilization of complementary probe (CP2) and the mediator thionine (Thi). Horseradish peroxidase (HRP) acted as a blocking reagent and signal enhancer. Following base pairing, the modified Pt-Pd nanowires were captured on the surface of the gold electrode. After addition of H 2 O 2 , the Pt-Pd nanowires and HRP both catalyzed the reduction of H 2 O 2 and promoted the electron transfer via the mediator Thi, resulting in an amplified electrochemical signal. The electrical signal, best measured at a working voltage of −200 mV (vs a SCE), is logarithmically related to the concentration of the M. pneu DNA in the 0.1 pM to 20 nM concentration range, and the detection limit (at an S/N ratio of 3) is 0.03 pM. The assay is robust, sensitive and specific. Conceivably, it is a cost-effective alternative to the established PCR method for the detection of M. pneu in clinical samples. (author)

  12. Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes.

    Science.gov (United States)

    Gong, Xue; Li, Jinfu; Zhou, Wenjiao; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-05-30

    Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Radioactive probes for human gene localisation by in situ hybridisation

    International Nuclear Information System (INIS)

    Fennell, S.J.

    1980-07-01

    Radioactive probes of high specific activity have been used for human gene localisation on metaphase chromosome preparations. Human 5S ribosomal RNA was used as a model system, as a probe for the localisation of human 5S ribosomal genes. 125 I-labelled mouse 5S ribosomal RNA was used to study the 5S ribosomal gene content and arrangement in families with translocations on the long arm of chromosome 1 close to or containing the 5S ribosomal RNA locus, by in situ hybridisation to human metaphase chromosomes from peripheral blood cultures. This confirmed the chromosomal assignment of 5S ribosomal genes to 1q 42-43. In situ hybridisation probes were also prepared from recombinant plasmids containing Xenopus laevis oocyte 5S or 28S/18S gene sequences to give [ 3 H]-labelled cRNA and [ 3 H]-labelled nick-translated plasmid DNA. Studies on the kinetics of hybridisation of plasmid probes with and without ribosomal gene sequences questioned the role of plasmid DNA for amplification of signal during gene localisation. Gene localisation was obtained with nick-translated plasmid DNA containing the 28S/18S ribosomal DNA insert after short exposure times, but poor results were obtained using a [ 3 H]-labelled cRNA probe transcribed from the plasmid with the 5S gene insert. (author)

  14. Co-isolation of in vivo 32P-labeled specific transcripts and DNA without phenol extraction of nuclease digestion

    International Nuclear Information System (INIS)

    Hayes, S.; Hayes, C.; Brand, L.

    1981-01-01

    A method is described for isolation and quantitation of specific intact transcripts, for which a hybridization probe is available, from 32 P-labeled bacterial cells. The RNA is extracted in the absence of R Nase activity by incorporating an inert, physically removable R Nase inhibitor throughout the spheroplasting, cell lysis, and pronase digestion steps. [/sup 32/P]RNA is separated from [ 32 P]DNA, without recourse to phenol extraction of DNase treatment, on a Cs 2 SO/sub 4-/HCONH 2 step gradient in which the precipitated RNA forms a sharp band. Specific transcripts are purified from [ 32 P]RNA by physical separation of the transcript and hybridization probe using gel-exclusion chromatography. The gentleness of this technique enables the co-isolation of DNA and can facilitate the analysis of covalently joined RNA-DNA replication intermediates

  15. Identification of the autotrophic denitrifying community in nitrate removal reactors by DNA-stable isotope probing.

    Science.gov (United States)

    Xing, Wei; Li, Jinlong; Cong, Yuan; Gao, Wei; Jia, Zhongjun; Li, Desheng

    2017-04-01

    Autotrophic denitrification has attracted increasing attention for wastewater with insufficient organic carbon sources. Nevertheless, in situ identification of autotrophic denitrifying communities in reactors remains challenging. Here, a process combining micro-electrolysis and autotrophic denitrification with high nitrate removal efficiency was presented. Two batch reactors were fed organic-free nitrate influent, with H 13 CO 3 - and H 12 CO 3 - as inorganic carbon sources. DNA-based stable-isotope probing (DNA-SIP) was used to obtain molecular evidence for autotrophic denitrifying communities. The results showed that the nirS gene was strongly labeled by H 13 CO 3 - , demonstrating that the inorganic carbon source was assimilated by autotrophic denitrifiers. High-throughput sequencing and clone library analysis identified Thiobacillus-like bacteria as the most dominant autotrophic denitrifiers. However, 88% of nirS genes cloned from the 13 C-labeled "heavy" DNA fraction showed low similarity with all culturable denitrifiers. These findings provided functional and taxonomical identification of autotrophic denitrifying communities, facilitating application of autotrophic denitrification process for wastewater treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. The use of AMPPD as an alternative substrate for AP-mediated detection of nonradiolabeled DNA probes in Eucalyptus saligna

    OpenAIRE

    De Moura Campos Pardini, M. I. [UNESP; Wolff, J. L C [UNESP; Lopes, C. R. [UNESP

    1993-01-01

    We present a non-radioactive alternative to Southern's (J. Mol. Biol. 98: 503-517, 1975) DNA-DNA hybridization technique. The use of AMPPD - Disodium 3-(4-Methoxyspiro {1,2-dioxetane-3,2'tricyclo[3.3.1.1(3,7)]decan}-4-yl)phyenyl phosphate as an alternative substrate for AP-mediated detection of digoxigenin-11 dUTP-labeled probes made possible the simple and nonhazardous reuse of blots. We used 0.8 % agarose gels containing 30 mug per lane of Eucalyptus saligna DNA, digested with Eco RI, elect...

  17. Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater.

    Science.gov (United States)

    Key, Katherine C; Sublette, Kerry L; Duncan, Kathleen; Mackay, Douglas M; Scow, Kate M; Ogles, Dora

    2013-01-01

    Although the anaerobic biodegradation of methyl tert -butyl ether (MTBE) and tert -butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13 C 5 -MTBE, 13 C 1 -MTBE (only methoxy carbon labeled), or 13 C 4 -TBA. 13 C-DNA and 12 C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert -butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13 C-labeled MTBE and TBA in situ and the 13 C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three 13 C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix , were only detected in the clone libraries where MTBE and TBA were fully labeled with 13 C, suggesting that they were involved in processing carbon from the tert -butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13 C. It is likely that members of this genus were secondary degraders cross-feeding on 13 C-labeled metabolites such as acetate.

  18. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    Science.gov (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  20. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

    International Nuclear Information System (INIS)

    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

    2012-01-01

    Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

  1. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    Science.gov (United States)

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Graphene–palladium nanowires based electrochemical sensor using ZnFe{sub 2}O{sub 4}–graphene quantum dots as an effective peroxidase mimic

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Weiyan; Yang, Hongmei; Ma, Chao; Ding, Ya-nan [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Ge, Shenguang [Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022 (China); Yu, Jinghua [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China); Yan, Mei, E-mail: chm_yanm@126.com [Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022 (China)

    2014-12-10

    Highlights: • The nanohybrid ZnFe{sub 2}O{sub 4}/GQDs was developed by assembling the GQDs on the ZnFe{sub 2}O{sub 4} through a photo-Fenton reaction. • The ZnFe{sub 2}O{sub 4}/GQDs exhibited higher peroxidase-like activity and better stability than each individual and HRP. • An electrochemical sensor was fabricated using ZnFe{sub 2}O{sub 4}/GQDs nanohybrid as a mimic enzymatic to detect DNA. • Graphene and Pd nanowires were modified on the glassy carbon electrode, which improved the electronic transfer rate. - Abstract: We proposed an electrochemical DNA sensor by using peroxidase-like magnetic ZnFe{sub 2}O{sub 4}–graphene quantum dots (ZnFe{sub 2}O{sub 4}/GQDs) nanohybrid as a mimic enzymatic label. Aminated graphene and Pd nanowires were successively modified on glassy carbon electrode, which improved the electronic transfer rate as well as increased the amount of immobilized capture ssDNA (S1). The nanohybrid ZnFe{sub 2}O{sub 4}/GQDs was prepared by assembling the GQDs on the surface of ZnFe{sub 2}O{sub 4} through a photo-Fenton reaction, which was not only used as a mimic enzyme but also as a carrier to label complementary ssDNA (S3). By synergistically integrating highly catalytically activity of nano-sized GQDs and ZnFe{sub 2}O{sub 4}, the nanohybrid possessed highly-efficient peroxidase-like catalytic activity which could produce a large current toward the reduction of H{sub 2}O{sub 2} for signal amplification. Thionine was used as an excellent electron mediator. Compared with traditional enzyme labels, the mimic enzyme ZnFe{sub 2}O{sub 4}/GQDs exhibited many advantages such as environment friendly and better stability. Under the optimal conditions, the approach provided a wide linear range from 10{sup −16} to 5 × 10{sup −9} M and low detection limit of 6.2 × 10{sup −17} M. The remarkable high catalytic capability could allow the nanohybrid to replace conventional peroxidase-based assay systems. The new, robust and convenient assay systems

  3. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

    Science.gov (United States)

    Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

    2004-01-01

    Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

  4. Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

    Science.gov (United States)

    Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

    2014-01-15

    Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

    Science.gov (United States)

    Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

    2015-06-15

    Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Label-free probing of genes by time-domain terahertz sensing

    International Nuclear Information System (INIS)

    Bolivar, P Haring; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Buettner, R

    2002-01-01

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed

  7. Label-free probing of genes by time-domain terahertz sensing

    Energy Technology Data Exchange (ETDEWEB)

    Bolivar, P Haring [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Brucherseifer, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Nagel, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Kurz, H [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Bosserhoff, A [Institut fuer Pathologie, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg (Germany); Buettner, R [Institut fuer Pathologie, Universitaetsklinikum Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn (Germany)

    2002-11-07

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

  8. DNA Probe for Lactobacillus delbrueckii

    OpenAIRE

    Delley, Michèle; Mollet, Beat; Hottinger, Herbert

    1990-01-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-l...

  9. In situ, accurate, surface-enhanced Raman scattering detection of cancer cell nucleus with synchronous location by an alkyne-labeled biomolecular probe.

    Science.gov (United States)

    Zhang, Jing; Liang, Lijia; Guan, Xin; Deng, Rong; Qu, Huixin; Huang, Dianshuai; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

    2018-01-01

    A surface-enhanced Raman scattering (SERS) method for in situ detection and analysis of the intranuclear biomolecular information of a cell has been developed based on a small, biocompatible, nuclear-targeting alkyne-tagged deoxyribonucleic acid (DNA) probe (5-ethynyl-2'-deoxyuridine, EDU) that can specially accumulate in the cell nucleus during DNA replications to precisely locate the nuclear region without disturbance in cell biological activities and functions. Since the specific alkyne group shows a Raman peak in the Raman-silent region of cells, it is an interior label to visualize the nuclear location synchronously in real time when measuring the SERS spectra of a cell. Because no fluorescent-labeled dyes were used for locating cell nuclei, this method is simple, nondestructive, non- photobleaching, and valuable for the in situ exploration of vital physiological processes with DNA participation in cell organelles. Graphical abstract A universal strategy was developed to accurately locate the nuclear region and obtain precise molecular information of cell nuclei by SERS.

  10. Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

    Science.gov (United States)

    Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2015-02-15

    Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Detecting the effects of toxic agents on spermatogenesis using DNA probes

    International Nuclear Information System (INIS)

    Hecht, N.B.

    1987-01-01

    Advances in the molecular biology of spermatogenesis suggest that DNA probes can be used to monitor the effects of toxic agents in male germ cells of mammals. Molecular hybridization analyses with DNA probes can provide a reproducible methodology capable of detecting changes ranging from massive deletions to single base pair substitutions in the genome of exposed individuals. A constantly increasing number of DNA probes that can be used to detect such alterations in human sperm DNA exist for both ubiquitously expressed proteins and for genes solely expressed in the testis. In this chapter, the currently available testicular stage-specific and/or cell type-specific DNA probes and the techniques by which they can be utilized in reproductive toxicology studies are discussed. The advantages, limitations, and future technological advances of this novel biological marker system for the human male reproductive system are also considered

  12. Gene probes : principles and protocols [Methods in molecular biology, v. 179

    National Research Council Canada - National Science Library

    Rapley, Ralph; Aquino de Muro, Marilena

    2002-01-01

    ... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

  13. Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling.

    Science.gov (United States)

    Sicoli, Giuseppe; Mathis, Gérald; Aci-Sèche, Samia; Saint-Pierre, Christine; Boulard, Yves; Gasparutto, Didier; Gambarelli, Serge

    2009-06-01

    Double electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes' positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.

  14. Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

    Directory of Open Access Journals (Sweden)

    Townsend Henrik J

    2005-11-01

    Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

  15. Peroxidase activity in root hairs of cress (lepidium sativum L.) Cytochemical localization and radioactive labelling of wall bound peroxidase

    International Nuclear Information System (INIS)

    Zaar, K.

    1979-01-01

    The ultrastructural localization of peroxidase activity in young, growing root hairs of cress (Lepidium sativum L.) after assay with 3,3'-diaminobenzidine is reported. Prominent peroxidase activity has been found in the dictyosomes and the associated vesicles, in ribosomes on ER-cisternae, as well as in the cell wall. On the basis of both ultrastructural and cytochemical evidence it is proposed that peroxidase in root hairs is synthesized on the ER- and within dictyosome cisternae packaged and transported in secretory vesicles and extruded into the cell wall particularily at the tip region of a root hair. The kinetic of Golgi apparatus mediated peroxidasesecretion was monitored by measuring the 55 Fe protoheme content of primary cell walls. Peroxidase secretion seems to be enhanced during stress incubation in destilled water. Secretory activity in root hairs is 20 times higher than in cells of the root body. (author)

  16. Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment.

    Science.gov (United States)

    Pavlekovic, Marko; Schmid, Markus C; Schmider-Poignee, Nadja; Spring, Stefan; Pilhofer, Martin; Gaul, Tobias; Fiandaca, Mark; Löffler, Frank E; Jetten, Mike; Schleifer, K-H; Lee, Natuschka M

    2009-08-01

    Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H(2)O(2) significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H(2)O(2

  17. Detection of hepatitis A virus by hybridization with single-stranded RNA probes

    International Nuclear Information System (INIS)

    Xi, J.; Estes, M.K.; Metcalf, T.G.

    1987-01-01

    An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

  18. Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA detection.

    Science.gov (United States)

    He, Yi; Huang, Guangming; Cui, Hua

    2013-11-13

    It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

  19. Fluorescent carbon nanoparticle-based lateral flow biosensor for ultrasensitive detection of DNA.

    Science.gov (United States)

    Takalkar, Sunitha; Baryeh, Kwaku; Liu, Guodong

    2017-12-15

    We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Wound-induced expression of horseradish peroxidase.

    Science.gov (United States)

    Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A

    1994-01-01

    Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

  1. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    Science.gov (United States)

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. The preparation of radioactivity labelled DNA

    International Nuclear Information System (INIS)

    Ballance, P.; Morgan, J.; McGregor, G.; Durkacz, B.

    1984-01-01

    The nick translation reaction, which uses the endonuclease enzyme to incorporate pieces of DNA into the genetic material of other organisms, is being used more and more in Molecular Biology research for the preparation of pure DNA labelled with 32 P. However results are presented which show that high radiation doses are received by the hands of nick translation workers. A Scheme of Work to reduce these doses is described. (author)

  3. Cu2+-labeled dansyl compounds as fluorescent and PET probes for imaging apoptosis.

    Science.gov (United States)

    Han, Junyan; Wang, Xukui; Yu, MeiXiang

    2016-11-15

    Compound DNSTT-Cu 2+ , a novel chelate of Cu 2+ with DOTA conjugated to a fluorescent dansyl fragment, is developed for imaging cell apoptosis. Apoptotic U-87MG cells could be selectively visualized by the fluorescence of DNSTT-Cu 2+ from cytoplasm of cells, confirmed by the fluorescence of apoptosis cells co-labeled with Alexa Fluor 568-labeled annexin V, a conventional probe for selectively labeling membranes of apoptosis cells. A radioactive 64 Cu 2 + analog, DNSTT- 64 Cu 2+ , was easily synthesized, providing a potential PET probe for imaging apoptosis in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

    International Nuclear Information System (INIS)

    Rene, Brigitte; Masliah, Gregoire; Zargarian, Loussine; Mauffret, Olivier; Fermandjian, Serge

    2006-01-01

    Summary 13 C, 15 N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments

  5. 32P-labeling test for DNA damage

    International Nuclear Information System (INIS)

    Randerath, K.; Reddy, M.V.; Gupta, R.C.

    1981-01-01

    Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32 P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, β-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [γ- 32 P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'- 32 P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyl-eneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10 5 DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding

  6. Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

    International Nuclear Information System (INIS)

    McDonnell, P.J.; McDonnell, J.M.; Kessis, T.; Green, W.R.; Shah, K.V.

    1987-01-01

    Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 35 S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association

  7. Electrochemiluminescent DNA sensor based on controlled Zn-mediated grafting of diazonium precursors.

    Science.gov (United States)

    Torréns, Mabel; Ortiz, Mayreli; Bejarano-Nosas, Diego; O'Sullivan, Ciara K

    2015-07-01

    Controlled Zn-mediated grafting of a thin layer of a diazonium salt was used to functionalise a carbon electrode with ruthenium(II)-tris-bipyridine (Ru)-labelled DNA for use as a capture probe in an electrochemiluminescent genosensor. A secondary reporter probe was labelled with a ferrocene (Fc) molecule, and in the presence of the single-stranded DNA target a genocomplex formed, where the Fc-label effectively quenched the electrochemiluminescence of the signal emitted from the Ru-label. The spacing of the labels for maximum sensitivity and minimum detection limit was optimised, and the signal reproducibility and stability of the method was established.

  8. Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

    International Nuclear Information System (INIS)

    Li, Dongyang; Ying, Yibin; Wu, Jian; Niessner, Reinhard; Knopp, Dietmar

    2013-01-01

    We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL −1 ). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immuno detection. (author)

  9. Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments.

    Directory of Open Access Journals (Sweden)

    Jingfeng Gao

    Full Text Available In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl density gradients for DNA-stable isotope probing (SIP experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays.

  10. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

    Science.gov (United States)

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

    2015-01-01

    The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

  11. Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing.

    Science.gov (United States)

    Cébron, Aurélie; Bodrossy, Levente; Chen, Yin; Singer, Andrew C; Thompson, Ian P; Prosser, James I; Murrell, J Colin

    2007-10-01

    A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.

  12. DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Brendel, M.; Faeth, W.W.; Toper, R.

    1975-01-01

    Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

  13. The use of radionuclide DNA probe technology for epidemiological studies of tegumentary leishmaniasis in Mato Grosso state, Brazil

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN), Belo Horizonte, MG (Brazil); Fernandes, Octavio [Fundacao Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical; Heub, Marcia; Fontes, Cor Jesus [Universidade Federal do Mato Grosso, Cuiaba, MT (Brazil). Hospital Universitario Julio Muller; Carvalho, Maria de Lourdes Ribeiro; Melo, Maria Norma de [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Parasitologia

    2005-10-15

    DNA hybridisation, using probes labelled with 32 P, was used to type Leishmania samples isolated from patients living in endemic areas of Mato Grosso State (Brazil), and clinically diagnosed as having tegumentary leishmaniasis. k DNA cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes were used. The results showed that L. braziliensis is the predominant group infecting human patients in the state. Sixty-eight samples were typed, 64 samples (94.1%) belonging to the L. braziliensis complex and only four (5.9%) belonging to the L. mexicana complex. Accurate identification of the Leishmania permits better orientation of the medical follow-up, since clinical manifestations may vary depending on the complex to which the parasite belongs. The epidemiological information furnished by the identification of the Leishmania in given endemic area is also essential for the design of appropriate control measures. (author)

  14. The use of radionuclide DNA probe technology for epidemiological studies of tegumentary leishmaniasis in Mato Grosso state, Brazil

    International Nuclear Information System (INIS)

    Andrade, Antero Silva Ribeiro de; Fernandes, Octavio; Heub, Marcia; Fontes, Cor Jesus; Carvalho, Maria de Lourdes Ribeiro; Melo, Maria Norma de

    2005-01-01

    DNA hybridisation, using probes labelled with 32 P, was used to type Leishmania samples isolated from patients living in endemic areas of Mato Grosso State (Brazil), and clinically diagnosed as having tegumentary leishmaniasis. k DNA cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes were used. The results showed that L. braziliensis is the predominant group infecting human patients in the state. Sixty-eight samples were typed, 64 samples (94.1%) belonging to the L. braziliensis complex and only four (5.9%) belonging to the L. mexicana complex. Accurate identification of the Leishmania permits better orientation of the medical follow-up, since clinical manifestations may vary depending on the complex to which the parasite belongs. The epidemiological information furnished by the identification of the Leishmania in given endemic area is also essential for the design of appropriate control measures. (author)

  15. Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

    International Nuclear Information System (INIS)

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping

    2013-01-01

    Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling

  16. Organophosphonate-based PNA-functionalization of silicon nanowires for label-free DNA detection.

    Science.gov (United States)

    Cattani-Scholz, Anna; Pedone, Daniel; Dubey, Manish; Neppl, Stefan; Nickel, Bert; Feulner, Peter; Schwartz, Jeffrey; Abstreiter, Gerhard; Tornow, Marc

    2008-08-01

    We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

  17. Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

    Science.gov (United States)

    Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

    2009-06-01

    Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that

  18. Characterization of Actinomyces with genomic DNA fingerprints and rRNA gene probes.

    Science.gov (United States)

    Bowden, G; Johnson, J; Schachtele, C

    1993-08-01

    Cellular DNA from 25 Actinomyces naeslundii and Actinomyces viscosus strains belonging to the 7 taxonomic clusters of Fillery et al. (1978) and several unclustered strains was obtained by enzymatic and N-lauroylsarcosine/guanidine isothiocyanate treatment of whole cells, followed by extraction of the nucleic acid. The DNA samples were digested with restriction endonucleases BamHI or PvuII, and agarose gel electrophoresis was used to obtain DNA fingerprints. The DNA fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for comparison between the taxonomic cluster strains and strains within clusters. Representative strains from each taxonomic cluster provided different BamHI DNA fingerprints and ribotype patterns with 3 to 9 distinct bands. Some strains within a cluster showed identical ribotype patterns with both endonucleases (A. naeslundii B120 and A. naeslundii B102 from cluster 3), while others showed the same pattern with BamHI but a different pattern with PvuII (A. naeslundii ATCC 12104 and 398A from cluster 5). A viscosus ATCC 15987 (cluster 7) and its parent strain T6 yielded identical fingerprint and ribotype patterns. The genomic diversity revealed by DNA fingerprinting and ribotyping demonstrates that these techniques, which do not require phenotypic expression, are suited for study of the oral ecology of the Actinomyces, and for epidemiological tracking of specific Actinomyces strains associated with caries lesions and sites of periodontal destruction.

  19. Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

    International Nuclear Information System (INIS)

    Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

    2002-01-01

    Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

  20. DNA-enhanced peroxidase-like activity of layered double hydroxide nanosheets and applications in H2O2 and glucose sensing.

    Science.gov (United States)

    Chen, Lijian; Sun, Kaifang; Li, Peipei; Fan, Xianzhong; Sun, Jianchao; Ai, Shiyun

    2013-11-21

    LDH nanosheets were obtained via continuous impaction and exfoliation by herring sperm DNA molecules using a constant vibration method. DNA-LDH nanohybrids were composed by electrostatic forces and they exhibited DNA-enhanced peroxidase-like activity. The morphology and structure of DNA-LDH nanohybrids were analyzed by transmission electron microscopy (TEM), selected-area electron diffraction (SAED), X-ray diffraction (XRD), and atomic force microscopy (AFM) characterization. On the basis of the high catalytic activity of DNA/CuAl-LDH nanosheets, a rapid, sensitive, and convenient approach was developed for colorimetric detection of H2O2 and blood glucose. This method can be potentially applied in medical diagnostics and biotechnology fields.

  1. Enhanced resolution of DNA restriction fragments: A procedure by two-dimensional electrophoresis and double-labeling

    International Nuclear Information System (INIS)

    Yi, M.; Au, L.C.; Ichikawa, N.; Ts'o, P.O.

    1990-01-01

    A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an x,y coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 32P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map

  2. Polyfluorophore Labels on DNA: Dramatic Sequence Dependence of Quenching

    Science.gov (United States)

    Teo, Yin Nah; Wilson, James N.

    2010-01-01

    We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynu-cleoside fluorophores (ODFs)—a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3′ end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern–Volmer constants (KSV) of between 2.1 × 104 and 4.3 × 105M−1, measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a KSV that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the π stack of fluorophores in the amplified quenching of fluorescence. PMID:19780115

  3. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  4. An Electrochemical DNA Biosensor for the Detection of Salmonella Using Polymeric Films and Electrochemical Labels

    Science.gov (United States)

    Diaz Serrano, Madeline

    Waterborne and foodborne diseases are one of the principal public health problems worldwide. Microorganisms are the major agents of foodborne illness: pathogens such as Salmonella, Campylobacter jejuni and Escherichia coli, and parasites such as cryptosporidium. The most popular methods to detect Salmonella are based on culture and colony counting methods, ELISA, Gel electrophoresis and the polymerase chain reaction. Conventional detection methods are laborious and time-consuming, allowing for portions of the food to be distributed, marketed, sold and eaten before the analysis is done and the problem even detected. By these reasons, the rapid, easy and portable detection of foodborne organisms will facilitate the disease treatment. Our particular interest is to develop a nucleic acid biosensor (NAB) for the detection of pathogenic microorganisms in food and water samples. In this research, we report on the development of a NAB prototype using a polymer modified electrode surface together with sequences of different lengths for the OmpC gene from Salmonella as probes and Ferrocene-labeled target (Fc-ssDNA), Ferrocene-labeled tri(ethylene glycol) (Fc-PEG) and Ruthenium-Ferrocene (Ru-Fe) bimetallic complex as an electrochemical labels. We have optimized several PS films and anchored nucleic acid sequences with different lengths at gold and carbon surfaces. Non contact mode AFM and XPS were used to monitor each step of the NAB preparation, from polymer modification to oligos hybridization (conventional design). The hybridization reaction was followed electrochemically using a Fc-ssDNA and Fc-PEG in solution taking advantage of the morphological changes generated upon hybridization. We observed a small current at the potential for the Fe oxidation without signal amplification at +296 mV vs. Ag/AgCl for the Fc-ssDNA strategy and a small current at +524 mV for the Fc-PEG strategy. The immobilization, hybridization and signal amplification of Biotin- OmpC Salmonella genes

  5. A universal DNA-based protein detection system.

    Science.gov (United States)

    Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

    2013-09-25

    Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

  6. Electrochemical DNA sandwich assay with a lipase label for attomole detection of DNA

    DEFF Research Database (Denmark)

    Ferapontova, Elena; Hansen, Majken Nørgaard; Saunders, Aaron Marc

    2010-01-01

    A fast and sensitive electrochemical lipase-based sandwich hybridization assay for detection of attomole levels of DNA has been developed. A combination of magnetic beads, used for pre-concentration and bioseparation of the analyte with a lipase catalyst label allowed detection of DNA with a limi...

  7. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi; PCR associado a hibridizacao com sondas radioativas de DNA para a identificacao de infeccao subclinica causada por Leishmania Chagasi

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Moreno, Elizabeth Castro [Fundacao Nacional de Saude, Belo Horizonte, MG (Brazil). Coordenacao Regional de Minas Gerais; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical

    2002-07-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with {sup 32} P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with {sup 32} P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  8. Design of 240,000 orthogonal 25mer DNA barcode probes.

    Science.gov (United States)

    Xu, Qikai; Schlabach, Michael R; Hannon, Gregory J; Elledge, Stephen J

    2009-02-17

    DNA barcodes linked to genetic features greatly facilitate screening these features in pooled formats using microarray hybridization, and new tools are needed to design large sets of barcodes to allow construction of large barcoded mammalian libraries such as shRNA libraries. Here we report a framework for designing large sets of orthogonal barcode probes. We demonstrate the utility of this framework by designing 240,000 barcode probes and testing their performance by hybridization. From the test hybridizations, we also discovered new probe design rules that significantly reduce cross-hybridization after their introduction into the framework of the algorithm. These rules should improve the performance of DNA microarray probe designs for many applications.

  9. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  10. Expression, purification and characterization of a peroxidase from ...

    African Journals Online (AJOL)

    Peroxidase is one of the key enzymes of the cellular antioxidant defense system, which is mostly involved in the reduction of hydrogen peroxide. Here, a peroxidase gene, named ThPOD1 was isolated from a cDNA library, which was generated from root tissue of Tamarix hispida that was exposed to 0.4 M NaCl. The cDNA ...

  11. Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

    Science.gov (United States)

    Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

    2018-04-17

    Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-11-01

    Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

  13. Unlabeled probes for the detection and typing of herpes simplex virus.

    Science.gov (United States)

    Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

    2007-10-01

    Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

  14. Applications of DNA-Stable Isotope Probing in Bioremediation Studies

    Science.gov (United States)

    Chen, Yin; Vohra, Jyotsna; Murrell, J. Colin

    DNA-stable isotope probing, a method to identify active microorganisms without the prerequisite of cultivation, has been widely applied in the study of microorganisms involved in the degradation of environmental pollutants. Recent advances and technique considerations in applying DNA-SIP in bioremediation are highlighted. A detailed protocol of a DNA-SIP experiment is provided.

  15. A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

    International Nuclear Information System (INIS)

    Hou, Sen; Tabaka, Marcin; Sun, Lili; Trochimczyk, Piotr; Kaminski, Tomasz S; Kalwarczyk, Tomasz; Zhang, Xuzhu; Holyst, Robert

    2014-01-01

    We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value. (letter)

  16. Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

    Science.gov (United States)

    Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

    2006-01-01

    We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

  17. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    Science.gov (United States)

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  18. Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair.

    Science.gov (United States)

    Qing, Taiping; He, Xiaoxiao; He, Dinggeng; Ye, Xiaosheng; Shangguan, Jingfang; Liu, Jinquan; Yuan, Baoyin; Wang, Kemin

    2017-08-15

    DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. In situ hybridization of phytoplankton using fluorescently labeled rRNA probes

    OpenAIRE

    Groben, R.; Medlin, Linda

    2005-01-01

    Fluorescently-labelled molecular probes were used to identify and characterise phytoplankton species using in situ hybridisation coupled with fluorescence microscopy and flow cytometry. The application of this technique is sometimes problematic, because of the many different species with which this method is to be used. Problems that may occur are: probe penetration versus maintanance of cell stability, strong autofluorescence and/or cell lost during the sample processing. Here we present a m...

  20. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  1. Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

    DEFF Research Database (Denmark)

    Yao, Danfeng; Kim, Junyoung; Yu, Fang

    2005-01-01

    at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA....../DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo...

  2. 2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    El-Yazbi, Amira F.; Loppnow, Glen R.

    2012-01-01

    Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

  3. DNA Diagnostics: Optical or by Electronics?

    KAUST Repository

    Khan, Hadayat Ullah

    2016-01-15

    In this paper, we very briefly review DNA biosensors based on optical and electrical detection principles, referring mainly to our past work applying both techniques but here using nearly identical sensor chip surface architectures, i.e., capture probe layers that were prepared based on a pulsed plasma deposition protocol for maleic anhydride and subsequent wet-chemical attachment of the amine-functionalized peptide nucleic acid (PNA) probe oligonucleotides. 15 mer DNA target strands, labeled with Cy5-chromophores that were attached at the 5’ end were used for surface plasmon optical detection and the same target DNA but without label was used in OTFT sensor-based detection where the mere charge density of the bound (hybridized) DNA molecules modulate the source-drain current. The sensing mechanisms and the detection limits of the devices are described in some detail. Both techniques allow for the monitoring of surface hybridization reactions, and offer the capacity to quantitatively discriminate between targets with different degrees of mismatched sequences.

  4. Electrogenerated chemiluminescence detection for deoxyribonucleic acid hybridization based on gold nanoparticles carrying multiple probes

    International Nuclear Information System (INIS)

    Wang Hui; Zhang Chengxiao; Li Yan; Qi Honglan

    2006-01-01

    A novel sensitive electrogenerated chemiluminescence (ECL) method for the detection deoxyribonucleic acid (DNA) hybridization based on gold nanoparticles carrying multiple probes was developed. Ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy) 2 (dcbpy)NHS) was used as a ECL label and gold nanoparticle as a carrier. Probe single strand DNA (ss-DNA) was self-assembled at the 3'-terminal with a thiol group to the surface of gold nanoparticle and covalently labeled at the 5'-terminal of a phosphate group with Ru(bpy) 2 (dcbpy)NHS and the resulting conjugate (Ru(bpy) 2 (dcbpy)NHS)-ss-DNA-Au, was taken as a ECL probe. When target analyte ss-DNA was immobilized on a gold electrode by self-assembled monolayer technique and then hybridized with the ECL probe to form a double-stranded DNA (ds-DNA), a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of the complementary sequence (target ss-DNA) in the range from 1.0 x 10 -11 to 1.0 x 10 -8 mol L -1 , and the linear regression equation was S = 57301 + 4579.6 lg C (unit of C is mol L -1 ). A detection limit of 5.0 x 10 -12 mol L -1 for target ss-DNA was achieved. The ECL signal generated from many reporters of ECL probe prepared is greatly amplified, compared to the convention scheme which is based on one reporter per hybridization event

  5. Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

    Science.gov (United States)

    Ahour, F; Shamsi, A

    2017-09-01

    Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

    Science.gov (United States)

    Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

    2005-08-01

    In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

  7. DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

    Science.gov (United States)

    Jameson, Eleanor; Taubert, Martin; Coyotzi, Sara; Chen, Yin; Eyice, Özge; Schäfer, Hendrik; Murrell, J Colin; Neufeld, Josh D; Dumont, Marc G

    2017-01-01

    Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13 C, 18 O, or 15 N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies

  8. A Chemical Probe that Labels Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Nao Hirata

    2014-03-01

    Full Text Available A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1] that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1 and ABCG2 (BCRP, both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

  9. Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

    Science.gov (United States)

    Ingianni, A; Floris, M; Palomba, P; Madeddu, M A; Quartuccio, M; Pompei, R

    2001-10-01

    Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories. Copyright 2001 Academic Press.

  10. DNA labeled during phosphonoacetate inhibition and following its reversal in herpesvirus infected cells

    International Nuclear Information System (INIS)

    Jacob, R.J.

    1984-01-01

    Human embryonic lung cells were pre-equilibrated with phosphonoacetate and 32 P orthophosphate label, then infected with phosphonoacetate-sensitive herpes simplex virus (HSV) type 1. Analyses of viral DNA produced in these cells showed the following. i) Viral DNA was synthesized in infected cells exposed to 100 μg of the drug per ml of medium but not in cells exposed to four-fold higher concentrations of the drug. ii) At 300 μg/ml a region of the DNA between 0.58 and 0.69 map units became transiently labeled, but the restriction endonuclease fragment containing these sequences migrated more slowly than the corresponding fragment from virion DNA. iii) Viral DNA extracted from infected cells 1.5 hours post drug withdrawal (300 μg/ml) was preferentially labeled in 2 regions of the genome mapping between 0.17 and 0.23 and 0.58-0.69 map units. This finding is in agreement with a report of Friedman et al. suggesting that HSV DNA contains two different sites if initiation. In addition a 4.8 x 10 6 molecular weight fragment was also preferentially labeled. This fragment could represent a smaller, aberrantly migrating fragment from the 0.17-0.27 map unit region of the DNA. iv) Viral DNA extracted from infected cells at longer intervals after drug withdrawal showed an increasing gradient of radioactivity progressively labeling the genome. These results are consistent with the hypothesis that viral DNA has at least two sites of initiation of DNA synthesis and that both sites are within the L component of the DNA. Alternatively, the results could be interpreted as two sites of localized synthesis (repair) that are detected at high concentrations of phosphonoacetate and immediately following reversal of inhibition of DNA synthesis. The results do not exclude the possibility that secondary sites in both L and S are utilized late in infection or in untreated cells. (Author)

  11. Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels.

    Science.gov (United States)

    Smith, L M; Sabnis, D D; Johnson, R P

    1987-04-01

    Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.

  12. Synthetic LNA/DNA nano-scaffolds for highly efficient diagnostics of nucleic acids and autoimmune antibodies

    DEFF Research Database (Denmark)

    Astakhova, Irina Kira

    2014-01-01

    ) strands and a series of fluorescent azides. The multiply labeled fluorescent LNA/DNA probes prepared herein generally display high binding affinity to complementary DNA/RNA, high quantum yields and, hence, high fluorescence brightness values. With the novel fluorescent probes, specific sensing...

  13. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  14. The field effect transistor DNA biosensor based on ITO nanowires in label-free hepatitis B virus detecting compatible with CMOS technology.

    Science.gov (United States)

    Shariati, Mohsen

    2018-05-15

    In this paper the field-effect transistor DNA biosensor for detecting hepatitis B virus (HBV) based on indium tin oxide nanowires (ITO NWs) in label free approach has been fabricated. Because of ITO nanowires intensive conductance and functional modified surface, the probe immobilization and target hybridization were increased strongly. The high resolution transmission electron microscopy (HRTEM) measurement showed that ITO nanowires were crystalline and less than 50nm in diameter. The single-stranded hepatitis B virus DNA (SS-DNA) was immobilized as probe on the Au-modified nanowires. The DNA targets were measured in a linear concentration range from 1fM to 10µM. The detection limit of the DNA biosensor was about 1fM. The time of the hybridization process for defined single strand was 90min. The switching ratio of the biosensor between "on" and "off" state was ~ 1.1 × 10 5 . For sensing the specificity of the biosensor, non-complementary, mismatch and complementary DNA oligonucleotide sequences were clearly discriminated. The HBV biosensor confirmed the highly satisfied specificity for differentiating complementary sequences from non-complementary and the mismatch oligonucleotides. The response time of the DNA sensor was 37s with a high reproducibility. The stability and repeatability of the DNA biosensor showed that the peak current of the biosensor retained 98% and 96% of its initial response for measurements after three and five weeks, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. A graphene oxide-based sensing platform for the label-free assay of DNA sequence and exonuclease activity via long range resonance energy transfer.

    Science.gov (United States)

    Jiang, Yixuan; Tian, Jianniao; Chen, Sheng; Zhao, Yanchun; Wang, Yuan; Zhao, Shulin

    2013-07-01

    Graphene oxide (GO) was introduced as an efficient quencher for label-free and sensitive detection of DNA. Probe DNA (pDNA) was mixed with ethidium bromide (EB) and graphene oxide (GO). The interaction between EB and GO led to the fluorescent quenching. Upon the recognition of the target, EB was intercalated into duplex DNA and kept away from GO, which significantly hindered the long range resonance energy transfer (LrRET) from EB to GO and, thus, increased the fluorescence of EB. The changes in fluorescent intensity produced a novel method for sensitivity, and specificity detection of the target. Based on the structure-switching of aptamers, this strategy could be conveniently extended for detection of other biomolecules, which had been demonstrated by the detection of exonuclease activity.

  16. Novel DNA sequence detection method based on fluorescence energy transfer

    International Nuclear Information System (INIS)

    Kobayashi, S.; Tamiya, E.; Karube, I.

    1987-01-01

    Recently the detection of specific DNA sequence, DNA analysis, has been becoming more important for diagnosis of viral genomes causing infections disease and human sequences related to inherited disorders. These methods typically involve electrophoresis, the immobilization of DNA on a solid support, hybridization to a complementary probe, the detection using labeled with /sup 32/P or nonisotopically with a biotin-avidin-enzyme system, and so on. These techniques are highly effective, but they are very time-consuming and expensive. A principle of fluorescene energy transfer is that the light energy from an excited donor (fluorophore) is transferred to an acceptor (fluorophore), if the acceptor exists in the vicinity of the donor and the excitation spectrum of donor overlaps the emission spectrum of acceptor. In this study, the fluorescence energy transfer was applied to the detection of specific DNA sequence using the hybridization method. The analyte, single-stranded DNA labeled with the donor fluorophore is hybridized to a probe DNA labeled with the acceptor. Because of the complementary DNA duplex formation, two fluorophores became to be closed to each other, and the fluorescence energy transfer was occurred

  17. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    Science.gov (United States)

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  18. Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

    Science.gov (United States)

    Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

    2015-12-15

    In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.

    OpenAIRE

    Noll, W W; Collins, M

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and th...

  20. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf; Weier, Jingly F.; Wang, Mei; Escudero, Tomas; Munne' , Santiago; Zitzelsberger, Horst F.; Weier, Heinz-Ulrich

    2009-01-30

    Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.

  1. Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

    Science.gov (United States)

    Zeppa, Pio; Sosa Fernandez, Laura Virginia; Cozzolino, Immacolata; Ronga, Valentina; Genesio, Rita; Salatiello, Maria; Picardi, Marco; Malapelle, Umberto; Troncone, Giancarlo; Vigliar, Elena

    2012-12-25

    The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society. Copyright © 2012 American Cancer Society.

  2. Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures

    International Nuclear Information System (INIS)

    Liu, Ting; Chen, Xian; Hong, Cheng-Yi; Xu, Xiao-Ping; Yang, Huang-Hao

    2014-01-01

    Electrochemiluminescence (ECL) integrates the advantages of electrochemical detection and chemiluminescent techniques. The method has received particular attention because it is highly sensitive and selective, has a wide linear range but low reagent costs. The use of nanomaterials with their unique physical and chemical properties has led to new kinds of biosensors that exhibit high sensitivity and stability. Compared to other nanomaterials, DNA nanostructures are more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption onto the electrode surface. We describe here a label-free and ultrasensitive ECL biosensor for detecting a cancer-associated microRNA at a femtomolar level. We have designed two auxiliary probes that cause the formation of a long-range self-assembly in the form of a μm-long 1-dimensional DNA concatamer. These can be used as carriers for signal amplification. The intercalation of the ECL probe Ru(phen) 3 2+ into the grooves of the concatamers leads to a substantial increase in ECL intensity. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of microRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis. (author)

  3. Detection of irradiation induced modifications in foodstuff DNA using 32p post-labelling

    International Nuclear Information System (INIS)

    Hoey, B.M.; Swallow, A.J.; Margison, G.P.

    1991-01-01

    DNA post-labelling has been used successfully to detect damage to DNA caused by a range of damaging agents. The assay results in a fingerprint of changes induced in DNA which might, in principle, be useful as a test for the detection of the irradiation of foods. The authors present their DNA extraction and 32 p post-labelling methods from chicken or cooked prawn samples and their analysis method (High Performance liquid chromatography). It's hoped that these results could form the basis of a test to detect if foods have been irradiated

  4. Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

    Science.gov (United States)

    Kjaersgård, I V; Jespersen, H M; Rasmussen, S K; Welinder, K G

    1997-03-01

    cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent

  5. Application of synthetic DNA probes to the analysis of DNA sequence variants in man

    International Nuclear Information System (INIS)

    Wallace, R.B.; Petz, L.D.; Yam, P.Y.

    1986-01-01

    Oligonucleotide probes provide a tool to discriminate between any two alleles on the basis of hybridization. Random sampling of the genome with different oligonucleotide probes should reveal polymorphism in a certain percentage of the cases. In the hope of identifying polymorphic regions more efficiently, we chose to take advantage of the proposed hypermutability of repeated DNA sequences and the specificity of oligonucleotide hybridization. Since, under appropriate conditions, oligonucleotide probes require complete base pairing for hybridization to occur, they will only hybridize to a subset of the members of a repeat family when all members of the family are not identical. The results presented here suggest that oligonucleotide hybridization can be used to extend the genomic sequences that can be tested for the presence of RFLPs. This expands the tools available to human genetics. In addition, the results suggest that repeated DNA sequences are indeed more polymorphic than single-copy sequences. 28 references, 2 figures

  6. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode

    Czech Academy of Sciences Publication Activity Database

    Němcová, Kateřina; Šebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-01-01

    Roč. 406, č. 24 (2014), s. 5843-5852 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GAP301/11/2076; GA AV ČR(CZ) IAA400040901 Institutional support: RVO:68081707 Keywords : Electrochemical analysis * Labeled probes * Osmium complex Subject RIV: BO - Biophysics Impact factor: 3.436, year: 2014

  7. Data Mining Empowers the Generation of a Novel Class of Chromosome-specific DNA Probes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Hui; Weier, Heinz-Ulrich G.; Kwan, Johnson; Wang, Mei; O' Brien, Benjamin

    2011-03-08

    Probes that allow accurate delineation of chromosome-specific DNA sequences in interphase or metaphase cell nuclei have become important clinical tools that deliver life-saving information about the gender or chromosomal make-up of a product of conception or the probability of an embryo to implant, as well as the definition of tumor-specific genetic signatures. Often such highly specific DNA probes are proprietary in nature and have been the result of extensive probe selection and optimization procedures. We describe a novel approach that eliminates costly and time consuming probe selection and testing by applying data mining and common bioinformatics tools. Similar to a rational drug design process in which drug-protein interactions are modeled in the computer, the rational probe design described here uses a set of criteria and publicly available bioinformatics software to select the desired probe molecules from libraries comprised of hundreds of thousands of probe molecules. Examples describe the selection of DNA probes for the human X and Y chromosomes, both with unprecedented performance, but in a similar fashion, this approach can be applied to other chromosomes or species.

  8. Coaxial atomic force microscope probes for dielectrophoresis of DNA under different buffer conditions

    Science.gov (United States)

    Tao, Yinglei; Kumar Wickramasinghe, H.

    2017-02-01

    We demonstrate a coaxial AFM nanoprobe device for dielectrophoretic (DEP) trapping of DNA molecules in Tris-EDTA (TE) and phosphate-buffered saline (PBS) buffers. The DEP properties of 20 nm polystyrene beads were studied with coaxial probes in media with different conductivities. Due to the special geometry of our DEP probe device, sufficiently high electric fields were generated at the probe end to focus DNA molecules with positive DEP. DEP trapping for both polystyrene beads and DNA molecules was quantitatively analyzed over the frequency range from 100 kHz to 50 MHz and compared with the Clausius-Mossotti theory. Finally, we discussed the negative effect of medium salinity during DEP trapping.

  9. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

    Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

  10. Concentrating and labeling genomic DNA in a nanofluidic array

    DEFF Research Database (Denmark)

    Marie, Rodolphe; Pedersen, Jonas Nyvold; Mir, Kalim U.

    2018-01-01

    , however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due...... to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final...

  11. Radioactive probes as diagnostic tools for rice tungro viruses

    International Nuclear Information System (INIS)

    Azzam, O.; Arboleda, M.; Reyes. J. de los

    1996-01-01

    Rice tungro bacilliform (RTBV) and rice tungro spherical viruses (RTSV) are the two viral components responsible for rice tungro disease which has seriously affected the irrigated rice ecosystem in Southeast Asia for the last 30 years. RTBV has an 8 Kb double-stranded DNA circular genome, and it is primarily responsible for induction of symptoms in infected plants. RTSV has a 12 kb single-stranded RNA genome. It does not induce any apparent symptoms in the infected plant, and it is transmitted by greenleafhopper. RTBV depends upon RTSV for its own transmission. The two viruses are limited to the vascular tissue of the rice plant and are present at a low titer. Most of the detection methods used for the identification of these viruses have relied on the virus protein properties and therefore, early detection of the virus activity was not possible. We were interested in evaluating tissue printing, dot blot, and southern techniques for early detection of virus nucleic acids in rice plant using radioactive and non radioactive probes. 32 P-labeled T7 or SP6 RNA polymerase transcripts complementary to the RTBV genome and RTSV coat protein genes were used as probes of the positive stand of both viruses. For nonradioactive probes, RTBV DNA genome was labeled using the ECL detection kit (Amersham). Preliminary results show that viral nucleic acids of RTBV and RTSV could be detected using both labelling systems. Non radioactive probes were comparable in their sensitivity to the radioactive probes. Less than 100 pg of viral DNA was detected in the dot-blot assays. More data will be presented to compare the efficiency and reliability of these two techniques in detecting early virus activity in the rice plant. (author)

  12. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    Directory of Open Access Journals (Sweden)

    Nattawut Yotapan

    2014-09-01

    Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  13. Selective detection of labeled DNA using an air-clad photonic crystal fiber

    DEFF Research Database (Denmark)

    Jensen, Jesper Bo Damm; Hoiby, P.E.; Pedersen, L.H.

    2004-01-01

    Demonstration of selective detection of fluorophore labeled DNA by hybridization inside the air holes of a photonic crystal fiber A laser exposes the fiber from the side and the emitted fluorescence tunnels into the core.......Demonstration of selective detection of fluorophore labeled DNA by hybridization inside the air holes of a photonic crystal fiber A laser exposes the fiber from the side and the emitted fluorescence tunnels into the core....

  14. Detection of experimental pancreas necrosis by DNA-ase labelled with radioiodine

    International Nuclear Information System (INIS)

    Tihanyi, Tibor; Duffek, Laszlo; Balint, Istvan; Flautner, Lajos

    1986-01-01

    Detection of pancreas necrosis was attempted in dog experiments. Strong association between pancreatic DNA-ase and actin molecules in vitro provided the theoretical basis for the procedure. Pancreatic DNA-ase labelled with 125 I was administered intravenously to dogs in which experimental pancreas necrosis was elicited. Accumulation ratio of radioactivity was above 20 in the necrotic pancreas whereas it varied between 1.6 and 5.6 in other tissues. After administration of 37 MBq 131 I labelled DNA-ase, accumulation of radioactivity could be clearly visualized in the necrotic portions of the removed pancreas by a gamma camera. The investigations will be extended in order to develop a clinically utilizable test. (L.E.)

  15. A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform.

    Science.gov (United States)

    Gao, Wenhua; Zhang, An; Chen, Yunsheng; Chen, Zixuan; Chen, Yaowen; Lu, Fushen; Chen, Zhanguang

    2013-11-15

    Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a "signal-on" ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg(2+). A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)3(2+) as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T-T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg(2+) and induce the stem-loop open, meanwhile the ECL "signal-on" was triggered. The peak sensitivity with the lowest detection limit of 0.1 nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Tyramine Hydrochloride Based Label-Free System for Operating Various DNA Logic Gates and a DNA Caliper for Base Number Measurements.

    Science.gov (United States)

    Fan, Daoqing; Zhu, Xiaoqing; Dong, Shaojun; Wang, Erkang

    2017-07-05

    DNA is believed to be a promising candidate for molecular logic computation, and the fluorogenic/colorimetric substrates of G-quadruplex DNAzyme (G4zyme) are broadly used as label-free output reporters of DNA logic circuits. Herein, for the first time, tyramine-HCl (a fluorogenic substrate of G4zyme) is applied to DNA logic computation and a series of label-free DNA-input logic gates, including elementary AND, OR, and INHIBIT logic gates, as well as a two to one encoder, are constructed. Furthermore, a DNA caliper that can measure the base number of target DNA as low as three bases is also fabricated. This DNA caliper can also perform concatenated AND-AND logic computation to fulfil the requirements of sophisticated logic computing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

    Science.gov (United States)

    de Ruiter, Mark V.; Overeem, Nico J.; Singhai, Gaurav; Cornelissen, Jeroen J. L. M.

    2018-05-01

    Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

  18. Catalytic nanocrystalline coordination polymers as an efficient peroxidase mimic for labeling and optical immunoassays

    International Nuclear Information System (INIS)

    Čunderlová, Veronika; Hlaváček, Antonín; Horňáková, Veronika; Peterek, Miroslav; Němeček, Daniel; Skládal, Petr; Hampl, Aleš; Eyer, Luděk

    2016-01-01

    We report that nanocrystalline Prussian blue of the type Fe 4 [Fe(CN) 6 ] 3 is a powerful peroxidase mimic for use in labeling of biomolecules. The cubic nanocrystals typically have a diameter of 15 nm and are capable of catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine in the presence of H 2 O 2 to form an intensively colored product with an absorption maximum at 662 nm. The determined pseudo turnover number is ∼20,000 s −1 which is the highest value reported for nanoparticles of a size comparable to common proteins. We also present a method for the biotinylation of the surface of these nanocrystals, and show their use in competitive bioaffinity based assays of biotin and human serum albumin. The limits of detection are 0.35 and 0.27 μg mL −1 , respectively. The results prove the applicability of coordination polymers for signal amplification and also their compatibility with the format of enzyme linked immunosorbent assays. (author)

  19. The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms.

    Science.gov (United States)

    Xu, Jian Z; Zhang, Jun L; Hu, Kai H; Zhang, Wei G

    2013-05-01

    Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  20. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  1. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  2. Decoding DNA labels by melting curve analysis using real-time PCR.

    Science.gov (United States)

    Balog, József A; Fehér, Liliána Z; Puskás, László G

    2017-12-01

    Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

  3. Probe Microscopic Studies of DNA Molecules on Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Kazuo Umemura

    2016-10-01

    Full Text Available Hybrids of DNA and carbon nanotubes (CNTs are promising nanobioconjugates for nanobiosensors, carriers for drug delivery, and other biological applications. In this review, nanoscopic characterization of DNA-CNT hybrids, in particular, characterization by scanning probe microscopy (SPM, is summarized. In many studies, topographical imaging by atomic force microscopy has been performed. However, some researchers have demonstrated advanced SPM operations in order to maximize its unique and valuable functions. Such sophisticated approaches are attractive and will have a significant impact on future studies of DNA-CNT hybrids.

  4. Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Youngblom, J.J. [California State University-Stanislaus, Turlock, CA (United States); Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

  5. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O

    2000-01-01

    Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown...... to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant...

  6. CONVERGENT SYNTHESIS AND EVALUATION OF 18F-LABELED AZULENIC COX2 PROBES FOR CANCER IMAGING

    Directory of Open Access Journals (Sweden)

    Donald D. Nolting

    2013-01-01

    Full Text Available The overall objectives of this research are to (i develop azulene-based PET probes and (ii image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel 18F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8+2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional 18F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an 18F labeling strategy that employed a much milder phosphate buffer. The 18F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for cancer imaging and deserves further

  7. Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder.

    Science.gov (United States)

    Wang, H F; Shortland, P; Park, M J; Grant, G

    1998-11-01

    In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6-S1 spinal cord segments and in the T13-L2 and L6-S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit-horseradish peroxidase was injected. Cholera toxin B subunit-horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV-VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin-horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit-horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I-horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit-horseradish peroxidase than with wheat germ agglutinin- or isolectin B4-horseradish peroxidase. In the L6-S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with

  8. Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

    Science.gov (United States)

    Ma, Jin-Liang; Yin, Bin-Cheng; Le, Huynh-Nhu; Ye, Bang-Ce

    2015-06-17

    We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

  9. Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

    Science.gov (United States)

    Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

    2016-12-15

    In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

    Science.gov (United States)

    Manzano, Marisa; Viezzi, Sara; Mazerat, Sandra; Marks, Robert S; Vidic, Jasmina

    2018-02-15

    Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results. Copyright © 2017. Published by Elsevier B.V.

  11. Expression, purification and characterization of a peroxidase from ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... from a cDNA library, which was generated from root tissue of Tamarix hispida that was exposed to ... enzymes, peroxidase (POD) plays an important role in .... ThPOD1 protein under various conditions, 3 month old T. hispida.

  12. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Malkondu, Sait [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Uyar, Pembegul [Selcuk University, Faculty of Science, Department of Biology, 42075 Konya (Turkey); Selcuk University, Advanced Technology Research and Application Center, Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey)

    2015-03-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope.

  13. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    International Nuclear Information System (INIS)

    Maltas, Esra; Malkondu, Sait; Uyar, Pembegul; Ozmen, Mustafa

    2015-01-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope

  14. Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols

    International Nuclear Information System (INIS)

    Hoy, C.A.; Lewis, E.D.; Schimke, R.T.

    1990-01-01

    The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [ 3 H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [ 3 H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [ 3 H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations

  15. The application of psoralens to the study of DNA structure, function and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Spielmann, Peter Hans [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1991-04-01

    A series of six nitroxide spin-labeled psoralens were designed, synthesized and tested as probes for DNA dynamics. The synthesis of these spin-labeled psoralen derivatives and their photoreactivity with double-stranded DNA fragments is described. The spin labels (nitroxides) were demonstrated to survive the uv irradiation required to bind the probe to the target DNA. EPR spectra of the photobound spin-labels indicate that they do not wobble with respect to the DNA on the time-scales investigated. The author has used psoralen modified DNA as a model for the study of DNA repair enzyme systems in human cell free extracts. He has shown that damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is "repair synthesis" and not an aberrant DNA synthesis reaction potentiated by deformation of the DNA by adducts. He has found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach he has obtained evidence that this in vitro system is capable of removing psoralen cross-links as well. Reported here are synthetic methods that make use of high intensity lasers coupled with HPLC purification to make homogeneous and very pure micromole quantities of furan-side monoadducted, cross-linked, and pyrone-side monoadducted DNA oligonucleotide. These molecules are currently being studied by NMR and X-ray crystallography. The application of the site-specifically psoralen modified oligonucleotide synthesized by these methods to the construction of substrates for the investigation of DNA repair is also discussed.

  16. Simple and label-free electrochemical impedance Amelogenin gene hybridization biosensing based on reduced graphene oxide.

    Science.gov (United States)

    Benvidi, Ali; Rajabzadeh, Nooshin; Mazloum-Ardakani, Mohammad; Heidari, Mohammad Mehdi; Mulchandani, Ashok

    2014-08-15

    The increasing desire for sensitive, easy, low-cost, and label free methods for the detection of DNA sequences has become a vital matter in biomedical research. For the first time a novel label-free biosensor for sensitive detection of Amelogenin gene (AMEL) using reduced graphene oxide modified glassy carbon electrode (GCE/RGO) has been developed. In this work, detection of DNA hybridization of the target and probe DNA was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The optimum conditions were found for the immobilization of probe on RGO surface and its hybridization with the target DNA. CV and EIS carried out in an aqueous solution containing [Fe(CN)6](3-/4-) redox pair have been used for the biosensor characterization. The biosensor has a wide linear range from 1.0×10(-20) to 1.0×10(-14)M with the lower detection limit of 3.2×10(-21)M. Moreover, the present electrochemical detection offers some unique advantages such as ultrahigh sensitivity, simplicity, and feasibility for apparatus miniaturization in analytical tests. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of RGO, which enhances the probe absorption and promotes direct electron transfer between probe and the electrode surface. This electrochemical DNA sensor could be used for the detection of specific ssDNA sequence in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.

    Science.gov (United States)

    Chen, Yin; Dumont, Marc G; Neufeld, Josh D; Bodrossy, Levente; Stralis-Pavese, Nancy; McNamara, Niall P; Ostle, Nick; Briones, Maria J I; Murrell, J Colin

    2008-10-01

    Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.

  18. DNA hybridization sensing for cytogenetic analysis

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dapra, Johannes; Brøgger, Anna Line

    2013-01-01

    are rearrangements between two chromosome arms that results in two derivative chromosomes having a mixed DNA sequence. The current detection method is a Fluorescent In situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the DNA sequences of two chromosomes involved...... in the translocation (Kwasny et al., 2012). We have developed a new double hybridization assay that allows for sorting of the DNA chromosomal fragments into separate compartment, moreover allowing for detection of the translocation. To detect the translocation it is necessary to determine that the two DNA sequences...... forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The first example of the translocation detection was presented on lab-on-a-disc using fluorescently labeled DNA fragments, representing the derivative chromosome (Brøgger et al., 2012). To allow...

  19. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  20. Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis

    Science.gov (United States)

    Enger, Lee; Joly, Sophie; Pujol, Claude; Simonson, Patricia; Pfaller, Michael; Soll, David R.

    2001-01-01

    Candida parapsilosis accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of C. parapsilosis genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of EcoRI/SalI-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies. PMID:11158125

  1. Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidases from lignin-degrading white rot fungi

    International Nuclear Information System (INIS)

    Bonnarme, P.; Jeffries, T.W.

    1990-01-01

    Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)-are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of 14 CO 2 from 14 C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of 14 CO 2 release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini

  2. Polypyrrole-poly(3,4-ethylenedioxythiophene)-Ag (PPy-PEDOT-Ag) nanocomposite films for label-free electrochemical DNA sensing.

    Science.gov (United States)

    Radhakrishnan, S; Sumathi, C; Umar, Ahmad; Jae Kim, Sang; Wilson, J; Dharuman, V

    2013-09-15

    The electrochemical DNA hybridization sensing of bipolymer polypyrrole and poly(3,4-ethylenedioxythiophene) (PPy-PEDOT) nanotubes functionalized with Ag nanoparticles has been investigated. The bipolymer nanotubes are prepared by simple chemical route and silver nanoparticles (Ag) further deposited over the PPy-PEDOT nanotubes to form PPy-PEDOT-Ag nanocomposite films. DNA labeled at 5'end using 6-mercapto-1-hexhane (HS-ssDNA) is immobilized on the PPy-PEDOT-Ag surface to form PPy-PEDOT-Ag-S-ssDNA and hybridization sensing is done in phosphate buffer. The presence of Ag nanoparticles (~28±5nm) well dispersed in the polymer composite with high surface area, high electrical conductivity and catalytic activity provides desirable microenvironment for the immobilization of probe DNA with controlled orientation leading to increased hybridization efficiency with target DNA. The morphological and structural characterizations by a scanning electron microscope (SEM) and X-ray diffraction (XRD) confirm the nanotube structure of composite polymer while Raman measurements indicate the efficient interactions between the PPy, PEDOT, Ag and HS-ssDNA. The sensor effectively discriminates different target DNA sequences with PPy-PEDOT-Ag-S-ssDNA substrate. The observed dynamic detection range is found between 1×10(-11)M and 1×10(-14)M with the lowest detection limit (3 σ/b) of 5.4×10(-15)M. This observed value is of higher sensitivity than that for MWCNT-Ag, PANi-Au, MWCNT-PPy-Au and PPy-PANi-Au composites reported previously. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Convergent synthesis and evaluation of {sup 18}F-labeled azulenic COX2 probes for cancer imaging

    Energy Technology Data Exchange (ETDEWEB)

    Nolting, Donald D.; Nickels, Michael; Tantawy, Mohammed N.; Yu, James Y. H.; Xie, Jingping [Department of Radiology, Institute of Imaging Science, Vanderbilt University, Nashville, TN (United States); Peterson, Todd E. [Department of Radiology, Institute of Imaging Science, Vanderbilt University, Nashville, TN (United States); Department of Physics and Astronomy, Vanderbilt University, Nashville, TN (United States); Crews, Brenda C. [Department of Chemistry, Vanderbilt University, Nashville, TN (United States); Vanderbilt Institute of Chemical Biology, Nashville, TN (United States); Marnett, Larry [Department of Chemistry, Vanderbilt University, Nashville, TN (United States); Vanderbilt Institute of Chemical Biology, Nashville, TN (United States); Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN (United States); Gore, John C. [Department of Radiology, Institute of Imaging Science, Vanderbilt University, Nashville, TN (United States); Department of Physics and Astronomy, Vanderbilt University, Nashville, TN (United States); Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN (United States); Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN (United States); Pham, Wellington, E-mail: wellington.pham@vanderbilt.edu [Department of Radiology, Institute of Imaging Science, Vanderbilt University, Nashville, TN (United States); Vanderbilt Institute of Chemical Biology, Nashville, TN (United States); Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN (United States); Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Department of Neuroscience, Vanderbilt University, Nashville, TN (United States)

    2013-01-03

    The overall objectives of this research are to (i) develop azulene-based positron emission tomography (PET) probes and (ii) image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel {sup 18}F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8 + 2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional {sup 18}F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an {sup 18}F labeling strategy that employed a much milder phosphate buffer. The {sup 18}F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for

  4. Labelling of the thymidine and deoxyctidine bases of DNA by (2-14C) deoxycytidine in cultured L1210 cells

    International Nuclear Information System (INIS)

    Karle, J.M.; Hoeraut, R.M.; Cysyk, R.L.

    1983-01-01

    Exposure of cultured L1210 cells to (2- 14 C) deoxycytidine and analysis of radioactivity incorporated into DNA-pyrimidines revealed that 2.7-5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus, the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favoured over successive phosphorlation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with (2- 14 C)-deoxycytidine. 3-deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (>=10 μM) increased the specific activity to DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (>= 10 μM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect. (author)

  5. Highly sensitive electrochemical detection of DNA hybridisation by coupling the chemical reduction of a redox label to the electrode reaction of a solution phase mediator.

    Science.gov (United States)

    Ngoensawat, Umphan; Rijiravanich, Patsamon; Somasundrum, Mithran; Surareungchai, Werasak

    2014-11-21

    We have described a highly sensitive method for detecting DNA hybridisation using a redox-labeled stem loop probe. The redox labels were poly(styrene-co-acrylic) (PSA) spheres of 454 nm diameter, modified by methylene blue (MB) deposited alternatively with poly(sodium 4-styrene sulphonate) (PSS) in a layer-by-layer process. Each PSA sphere carried approx. 3.7 × 10(5) molecules of MB, as determined optically. DIG-tagged stem loop probes were immobilised on screen printed electrodes bearing anti-DIG antibodies. Binding with the target enabled straightening of the stem loop, which made attachment to the MB-coated PSA spheres possible. For measuring the current from the direct reduction of MB by differential pulse voltammetry, a 30 mer DNA target common to 70 strains of Escherichia coli was calibrated across the range 1.0 fM to 100 pM (gradient = 3.2 × 10(-8) A (log fM)(-1), r(2) = 0.95, n = 60), with an LOD of ∼58 fM. By using Fe(CN)6(3-/4-) as a solution phase mediator for the MB reduction, we were able to lower the LOD to ∼39 aM (gradient = 5.95 × 10(-8) A (log aM)(-1), r(2) = 0.96, n = 30), which corresponds to the detection of 0.76 ag (∼50 molecules) in the 2 μL analyte sample. We hypothesise that the lowering of the LOD was due to the fact that not all the MB labels were able to contact the electrode surface.

  6. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.; Suzuki, M.; Adachi, S.

    1983-01-01

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  7. Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly

    DEFF Research Database (Denmark)

    Jakobsen, Ulla; Vogel, Stefan

    2016-01-01

    Assays for mismatch discrimination and detection of single nucleotide variations by hybridization-controlled assembly of liposomes, which do not require tedious surface chemistry, are versatile for both DNA and RNA targets. We report herein a comprehensive study on different DNA and LNA (locked...... assay in the context of mismatch discrimination and SNP detection are presented. The advantages of membrane-anchored LiNA-probes compared to chemically attached probes on solid nanoparticles (e.g. gold nanoparticles) are described. Key functionalities such as non-covalent attachment of LiNA probes...... without the need for long spacers and the inherent mobility of membrane-anchored probes in lipid-bilayer membranes will be described for several different probe designs....

  8. Gold surface supported spherical liposome-gold nano-particle nano-composite for label free DNA sensing.

    Science.gov (United States)

    Bhuvana, M; Narayanan, J Shankara; Dharuman, V; Teng, W; Hahn, J H; Jayakumar, K

    2013-03-15

    Immobilization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposome-gold nano-particle (DOPE-AuNP) nano-composite covalently on 3-mercaptopropionic acid (MPA) on gold surface is demonstrated for the first time for electrochemical label free DNA sensing. Spherical nature of the DOPE on the MPA monolayer is confirmed by the appearance of sigmoidal voltammetric profile, characteristic behavior of linear diffusion, for the MPA-DOPE in presence of [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+) redox probes. The DOPE liposome vesicle fusion is prevented by electroless deposition of AuNP on the hydrophilic amine head groups of the DOPE. Immobilization of single stranded DNA (ssDNA) is made via simple gold-thiol linkage for DNA hybridization sensing in the presence of [Fe(CN)(6)](3-/4-). The sensor discriminates the hybridized (complementary target hybridized), un-hybridized (non-complementary target hybridized) and single base mismatch target hybridized surfaces sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 0.1×10(-12)M. Cyclic voltammetry (CV), electrochemical impedance (EIS), differential pulse voltammetry (DPV) and quartz crystal microbalance (QCM) techniques are used for DNA sensing on DOPE-AuNP nano-composite. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet-Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE, AuNP and ssDNA. The results indicate the presence of an intact and well defined spherical DOPE-AuNP nano-composite on the gold surface. The method could be applied for fabrication of the surface based liposome-AuNP-DNA composite for cell transfection studies at reduced reagents and costs. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Gold nanoparticle-based probes for the colorimetric detection of Mycobacterium avium subspecies paratuberculosis DNA.

    Science.gov (United States)

    Ganareal, Thenor Aristotile Charles S; Balbin, Michelle M; Monserate, Juvy J; Salazar, Joel R; Mingala, Claro N

    2018-02-12

    Gold nanoparticle (AuNP) is considered to be the most stable metal nanoparticle having the ability to be functionalized with biomolecules. Recently, AuNP-based DNA detection methods captured the interest of researchers worldwide. Paratuberculosis or Johne's disease, a chronic gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), was found to have negative effect in the livestock industry. In this study, AuNP-based probes were evaluated for the specific and sensitive detection of MAP DNA. AuNP-based probe was produced by functionalization of AuNPs with thiol-modified oligonucleotide and was confirmed by Fourier-Transform Infrared (FTIR) spectroscopy. UV-Vis spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize AuNPs. DNA detection was done by hybridization of 10 μL of DNA with 5 μL of probe at 63 °C for 10 min and addition of 3 μL salt solution. The method was specific to MAP with detection limit of 103 ng. UV-Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results, respectively, with no observed particle growth. This study therefore reports an AuNP-based probes which can be used for the specific and sensitive detection of MAP DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

    International Nuclear Information System (INIS)

    Takahashi, Norio; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

    1990-05-01

    We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that use of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than use of DNA:DNA heteroduplexes, because preparation of RNA probes is easier than that of DNA probes. Three different 32 P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of a mismatch(es) was detected as a difference in the mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and three thalassemic human β-globin genes. The results from experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was undertaken in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for detecting heritable variation and should be a powerful tool for detecting fresh mutations in DNA, which occur outside the known restriction sites. (author)

  11. Probe DNA-Cisplatin Interaction with Solid-State Nanopores

    Science.gov (United States)

    Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

    2014-03-01

    Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

  12. A repetitive probe for FISH analysis of bovine interphase nuclei

    Directory of Open Access Journals (Sweden)

    Cribiu Edmond

    2000-03-01

    Full Text Available Abstract The purpose of this study was to generate repetitive DNA sequence probes for the analysis of interphase nuclei by fluorescent in situ hybridisation (FISH. Such probes are useful for the diagnosis of chromosomal abnormalities in bovine preimplanted embryos. Of the seven probes (E1A, E4A, Ba, H1A, W18, W22, W5 that were generated and partially sequenced, five corresponded to previously described Bos taurus repetitive DNA (E1A, E4A, Ba, W18, W5, one probe (W22 shared no homology with other DNA sequences and one (H1A displayed a significant homology with Rattus norvegicus mRNA for secretin receptor transmembrane domain 3. Fluorescent in situ hybridisation was performed on metaphase bovine fibroblast cells and showed that five of the seven probes hybridised most centromeres (E1A, E4A, Ba, W18, W22, one labelled the arms of all chromosomes (W5 and the H1A probe was specific to three chromosomes (ch14, ch20, and ch25. Moreover, FISH with H1A resulted in interpretable signals on interphase nuclei in 88% of the cases, while the other probes yielded only dispersed overlapping signals.

  13. Evaluation of the Gen-Probe DNA probe for the detection of legionellae in culture

    International Nuclear Information System (INIS)

    Edelstein, P.H.

    1986-01-01

    A commercial DNA probe kit designed to detect rRNA from legionellae was evaluated for its ability to correctly discriminate between legionellae and non-legionellae taken from culture plates. The probe kit, made by the Gen-Probe Corp. (San Diego, Calif.), was radiolabeled with 125 I, and probe bacterial RNA hybridization, detected in a simple one-tube system hybridization assay, was quantitated with a gamma counter. A total of 156 Legionella sp. strains were tested, of which 125 were Legionella pneumophila and the remainder were strains from 21 other Legionella spp. A total of 106 gram-negative non-legionellae, isolated from human respiratory tract (81%) and other body site (19%) specimens, were also tested; 14 genera and 28 species were represented. The probe easily distinguished all of the legionellae from the non-legionellae. The average legionellae/non-legionellae hybridization ratio was 42:1, and the lowest ratio was 2:1; a minor modification in the procedure increased the lowest ratio to 5:1. In addition to correctly identifying all Legionella species, the probe was able to separate some of the various species of Legionella. L. pneumophila strains hybridized more completely to the probe than did the other Legionella spp.; L. wadsworthii and L. oakridgensis hybridized only about 25% of the probe relative to L. pneumophila. Some strains of phenotypically identified L. pneumophila had much lower hybridization to the probe than other members of the species and may represent a new Legionella species. The simplicity of the technique and specificity of the probe make it a good candidate for confirming the identity of legionellae in culture

  14. Specific identification of human papillomavirus type in cervical smears and paraffin sections by in situ hybridization with radioactive probes: a preliminary communication

    International Nuclear Information System (INIS)

    Gupta, J.; Gendelman, H.E.; Naghashfar, Z.; Gupta, P.; Rosenshein, N.; Sawada, E.; Woodruff, J.D.; Shah, K.

    1985-01-01

    Cervical Papanicolaou smears and paraffin sections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using 35 S-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two 35 S-labeled nucleotides. Paraffin sections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material

  15. Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid

    International Nuclear Information System (INIS)

    Jellinck, P.H.; Newbold, R.R.; McLachlan, J.A.

    1991-01-01

    Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from [2-3H]estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from [2-3H]estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid

  16. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  17. A fluorescent DNA based probe for Hg(II) based on thymine-Hg(II)-thymine interaction and enrichment via magnetized graphene oxide.

    Science.gov (United States)

    Li, Meng-Ke; Hu, Liu-Yin; Niu, Cheng-Gang; Huang, Da-Wei; Zeng, Guang-Ming

    2018-03-03

    The authors describe a fluorometric assay for the determination of Hg(II). A naphthalimide derivative is used as a label for a thymine (T) rich ssDNA, and graphene oxide magnetized with Fe 3 O 4 nanoparticles acts as a quencher and preconcentrators. In the absence of Hg(II), the labeled ssDNA does not separate from the magnetized graphene oxide. As a result, fluorescence is fully quenched. In the presence of Hg(II), a T-Hg(II)-T link is formed dues to the highly affinity between T and Hg(II). Hence, fluorescence is restored. The assay has a linear response in the 1.0 to 10.0 nM Hg(II) concentration range, and a 0.65 nM detection limit. The method is selective and sensitive. It was applied to the analysis of spiked environmental water samples, and data agreed well with those obtained by atomic fluorescence spectrometry. Graphical abstract Strategy of a fluorescent probe for detecting Hg(II). The method has a 0.65 nM detection limit and is selective. MGO: magnetized graphene oxide, AHN: a fluorescent derivative of naphthalimide.

  18. Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

    International Nuclear Information System (INIS)

    Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

    1996-01-01

    Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

  19. Detection of Hepatitis B virus DNA and Hepatitis δ virus RNA

    International Nuclear Information System (INIS)

    Smedile, A.; Chiaberge, E.; Brunetto, M.R.; Negro, F.; Baldi, M.; Lavarini, C.; Maran, E.

    1987-01-01

    The recent availability of DNA probes of the Hepatitis B Virus DNA (HBV-DNA) and of Hepatitis Delta Virus RNA (HDV-RNA) allows the application of nucleic acid hybridization techniques to solve a variety of clinical problems. DNA probes of HBV-DNA and HDV-RNA are labeled by nick translation using 32 P or biotinylated nucleotides and hybridized to filters containing test nucleic acids. Complementary sequences are identified and the degree of blackening of the film at autoradiography or the enzymatic staining of the filter is proportional to the amount of viral nucleic acid hybridized to the probe and present in the sample. These procedures allow rapid examination of multiple specimens and are sensitive and reproducible. Viral nucleic acids can be measured quantitatively and their quantity correlates with the infectivity of sera titered in experimentally infected animals

  20. Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

    Directory of Open Access Journals (Sweden)

    Zach Hensel

    Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

  1. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    Science.gov (United States)

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  2. DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae

    Directory of Open Access Journals (Sweden)

    P. D. Ready

    1991-03-01

    Full Text Available Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

  3. The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

    International Nuclear Information System (INIS)

    Xi Dong; Luo Xiaoping; Lu Qianghua; Yao Kailun; Liu Zuli; Ning Qin

    2008-01-01

    Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method

  4. Molecular cloning and characterization of a new peroxidase gene ...

    African Journals Online (AJOL)

    length cDNA of O.violaceus peroxidase gene (OvRCI, GenBank. Acc. No. AY428037) was 1220 bp and contained an 1128 bp open reading frame encoding a protein of 375 amino acids. Homology analysis and molecular modeling revealed that ...

  5. DNA-labeled micro- and nanoparticles: a new approach to study contaminant transport in the subsurface

    Science.gov (United States)

    McNew, C.; Wang, C.; Kocis, T. N.; Murphy, N. P.; Dahlke, H. E.

    2017-12-01

    Though our understanding of contaminant behavior in the subsurface has improved, our ability to measure and predict complex contaminant transport pathways at hillslope to watershed scales is still lacking. By utilizing bio-molecular nanotechnology developed for nano-medicines and drug delivery, we are able to produce DNA-labeled micro- and nanoparticles for use in a myriad of environmental systems. Control of the fabrication procedure allows us to produce particles of custom size, charge, and surface functionality to mimic the transport properties of the particulate contaminant or colloid of interest. The use of custom sequenced DNA allows for the fabrication of an enormous number of unique particle labels (approximately 1.61 x 1060 unique sequences) and the ability to discern between varied spatial and temporal applications, or the transport effect of varied particle size, charge, or surface properties. To date, this technology has been utilized to study contaminant transport from lab to field scales, including surface and open channel flow applications, transport in porous media, soil retention, and even subglacial flow pathways. Here, we present the technology for production and detection of the DNA-labeled particles along with the results from a current hillslope study at the Sierra Foothills Research and Extension Center (SFREC). This field study utilizes spatial and temporal variations in DNA-labeled particle applications to identify subsurface pollutant transport pathways through the four distinct soil horizons present at the SFREC site. Results from this and previous studies highlight the tremendous potential of the DNA-labeled particle technology for studying contaminant transport through the subsurface.

  6. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique

    International Nuclear Information System (INIS)

    Noll, W.W.; Collins, M.

    1987-01-01

    Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. The authors have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. They have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases

  7. Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

    Science.gov (United States)

    Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

    2018-05-09

    The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

  8. Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

    Science.gov (United States)

    Wang, Feng; Liu, Juewen

    2014-05-01

    Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate

  9. Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

    Science.gov (United States)

    Najafzadeh, M J; Vicente, V A; Feng, Peiying; Naseri, A; Sun, Jiufeng; Rezaei-Matehkolaei, A; de Hoog, G S

    2018-03-05

    The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

  10. Asynchronous Magnetic Bead Rotation (AMBR Microviscometer for Label-Free DNA Analysis

    Directory of Open Access Journals (Sweden)

    Yunzi Li

    2014-03-01

    Full Text Available We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

  11. Probing the DNA Structural Requirements for Facilitated Diffusion

    Science.gov (United States)

    2015-01-01

    DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein. PMID:25495964

  12. Identification of metabolically active bacteria in the gut of the generalist Spodoptera littoralis via DNA stable isotope probing using 13C-glucose.

    Science.gov (United States)

    Shao, Yongqi; Arias-Cordero, Erika M; Boland, Wilhelm

    2013-11-13

    Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The

  13. Templated Chemistry for Sequence-Specific Fluorogenic Detection of Duplex DNA

    Science.gov (United States)

    Li, Hao; Franzini, Raphael M.; Bruner, Christopher; Kool, Eric T.

    2015-01-01

    We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine, designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of ca. 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably, demonstrating high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and may allow detection of double stranded DNA, at least for homopurine-homopyrimidine target sites, under native, non-disturbing conditions. PMID:20859985

  14. The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review

    Directory of Open Access Journals (Sweden)

    Jahwarhar Izuan Abdul Rashid

    2017-11-01

    Full Text Available In recent years, electrochemical deoxyribonucleic acid (DNA sensor has recently emerged as promising alternative clinical diagnostic devices especially for infectious disease by exploiting DNA recognition events and converting them into an electrochemical signal. This is because the existing DNA diagnostic method possesses certain drawbacks such as time-consuming, expensive, laborious, low selectivity and sensitivity. DNA immobilization strategies and mechanism of electrochemical detection are two the most important aspects that should be considered before developing highly selective and sensitive electrochemical DNA sensor. Here, we focus on some recent strategies for DNA probes immobilization on the surface of electrochemical transducer such as adsorption, covalent bonding and Avidin/Streptavidin-Biotin interaction on the electrode surface for specific interaction with its complementary DNA target. A numerous approach for DNA hybridization detection based electrochemical technique that frequently used including direct DNA electrochemical detection and label based electrochemical (redox-active indicator, enzyme label and nanoparticles were also discussed in aiming to provide general guide for the design of electrochemical DNA sensor. We also discussed the challenges and suggestions to improve the application of electrochemical DNA sensor at point-care setting. Keywords: Electrochemical DNA sensor, DNA immobilization, DNA hybridization, Electrochemical mechanism

  15. Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin.

    Science.gov (United States)

    Cobb, C E; Hustedt, E J; Beechem, J M; Beth, A H

    1993-01-01

    An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional

  16. Investigation of paternity establishing without the putative father using hypervariable DNA probes.

    Science.gov (United States)

    Yokoi, T; Odaira, T; Nata, M; Sagisaka, K

    1990-09-01

    Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

  17. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  18. Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNA

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Nes, Ingolf F.; Wells, Robert D.

    1976-01-01

    Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100–200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced...

  19. Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label.

    Science.gov (United States)

    Liu, Ke; Pan, Dun; Wen, Yanqin; Zhang, Honglu; Chao, Jie; Wang, Lihua; Song, Shiping; Fan, Chunhai; Shi, Yongyong

    2018-02-01

    The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high-efficiency hybridizable DNA origami-based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof-of-concept, five genotype B and six genotype C are detected in 11 HBV-infected patients' blood DNA samples of Han Chinese population in the single-blinded test. The AFM image-based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Intra-albumin migration of bound fatty acid probed by spin label ESR

    International Nuclear Information System (INIS)

    Gurachevsky, Andrey; Shimanovitch, Ekaterina; Gurachevskaya, Tatjana; Muravsky, Vladimir

    2007-01-01

    Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains

  1. Silver-gold core-shell nanoparticles containing methylene blue as SERS labels for probing and imaging of live cells

    International Nuclear Information System (INIS)

    Guo, X.; Guo, Z.; Jin, Y.; Liu, Z.; Zhang, W.; Huang, D.

    2012-01-01

    We report on silver-gold core-shell nanostructures that contain Methylene Blue (MB) at the gold/x96silver interface. They can be used as reporter molecules in surface-enhanced Raman scattering (SERS) labels. The labels are stable and have strong SERS activity. TEM imaging revealed that these nanoparticles display bright and dark stripe structures. In addition, these labels can act as probes that can be detected and imaged through the specific Raman signatures of the reporters. We show that such SERS probes can identify cellular structures due to enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the core-shell nanostructures. They also provide structural information on the cellular environment as demonstrated for these nanoparticles as new SERS-active and biocompatible substrates for imaging of live cells. (author)

  2. Single Molecule Screening of Disease DNA Without Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

    2006-01-01

    The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

  3. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

    2011-05-13

    We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  4. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    International Nuclear Information System (INIS)

    Ali, M E; Hashim, U; Mustafa, S; Che Man, Y B; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-01-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 0 C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μg ml -1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  5. Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions

    Science.gov (United States)

    Kemp, David J.; Smith, Donald B.; Foote, Simon J.; Samaras, N.; Peterson, M. Gregory

    1989-04-01

    The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.

  6. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    Science.gov (United States)

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  7. Multi-Probe Based Artificial DNA Encoding and Matching Classifier for Hyperspectral Remote Sensing Imagery

    Directory of Open Access Journals (Sweden)

    Ke Wu

    2016-08-01

    Full Text Available In recent years, a novel matching classification strategy inspired by the artificial deoxyribonucleic acid (DNA technology has been proposed for hyperspectral remote sensing imagery. Such a method can describe brightness and shape information of a spectrum by encoding the spectral curve into a DNA strand, providing a more comprehensive way for spectral similarity comparison. However, it suffers from two problems: data volume is amplified when all of the bands participate in the encoding procedure and full-band comparison degrades the importance of bands carrying key information. In this paper, a new multi-probe based artificial DNA encoding and matching (MADEM method is proposed. In this method, spectral signatures are first transformed into DNA code words with a spectral feature encoding operation. After that, multiple probes for interesting classes are extracted to represent the specific fragments of DNA strands. During the course of spectral matching, the different probes are compared to obtain the similarity of different types of land covers. By computing the absolute vector distance (AVD between different probes of an unclassified spectrum and the typical DNA code words from the database, the class property of each pixel is set as the minimum distance class. The main benefit of this strategy is that the risk of redundant bands can be deeply reduced and critical spectral discrepancies can be enlarged. Two hyperspectral image datasets were tested. Comparing with the other classification methods, the overall accuracy can be improved from 1.22% to 10.09% and 1.19% to 15.87%, respectively. Furthermore, the kappa coefficient can be improved from 2.05% to 15.29% and 1.35% to 19.59%, respectively. This demonstrated that the proposed algorithm outperformed other traditional classification methods.

  8. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    DEFF Research Database (Denmark)

    Sørensen, Karina; Andersen, Paal; Larsen, Lars

    2008-01-01

    The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim...... of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA...... analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total...

  9. Molecular studies of fibroblasts transfected with hepatitis B virus DNA

    International Nuclear Information System (INIS)

    Chen, M.L.; Hood, A.; Thung, S.N.; Gerber, M.A.

    1987-01-01

    Two subclones (D7 and F8) derived from an NIH 3T3 mouse fibroblast cell line after transfection with hepatitis B virus (HBV) genomes, secreted significantly different amounts of HBsAg and HBeAg. DNA extracted from the subclones revealed only integrated and no extrachromosomal HBV DNA sequences as determined by the Southern blot technique with a /sup 32/P-labeled full length HBV DNA probe. The amount and integration sites of HBV sequences were significantly different in the two subclones. HBV DNA sequences coding for HBsAg and HBcAg were detected by alkaline phosphatase-conjugated, single-stranded synthetic gene-specific oligonucleotide probes revealing a larger number of copies in D7 DNA than in F8 DNA. Using a biotinylated probe for in situ hybridization, HBV DNA was found in the nuclei of all D7 cells with predominant localization to a single chromsome, but only in 10-20% of F8 cells. These observations demonstrate different integration patterns of HBV and DNA in two subclones derived from a transfected cell line and suggest that the amount of integrated HBV DNA is proportional to the amount of HBV antigens produced

  10. Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst.

    Science.gov (United States)

    Pelossof, Gilad; Tel-Vered, Ran; Elbaz, Johann; Willner, Itamar

    2010-06-01

    The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme is assembled on Au electrodes. It reveals bioelectrocatalytic properties and electrocatalyzes the reduction of H(2)O(2). The bioelectrocatalytic functions of the hemin/G-quadruplex DNAzyme are used to develop electrochemical sensors that follow the activity of glucose oxidase and biosensors for the detection of DNA or low-molecular-weight substrates (adenosine monophosphate, AMP). Hairpin nucleic structures that include the G-quadruplex sequence in a caged configuration and the nucleic acid sequence complementary to the analyte DNA, or the aptamer sequence for AMP, are immobilized on Au-electrode surfaces. In the presence of the DNA analyte, or AMP, the hairpin structures are opened, and the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme structures are generated on the electrode surfaces. The bioelectrocatalytic cathodic currents generated by the functionalized electrodes, upon the electrochemical reduction of H(2)O(2), provide a quantitative measure for the detection of the target analytes. The DNA target was analyzed with a detection limit of 1 x 10(-12) M, while the detection limit for analyzing AMP was 1 x 10(-6) M. Methods to regenerate the sensing surfaces are presented.

  11. Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

    Science.gov (United States)

    Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

    2018-04-01

    ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

  12. Effect of structure variation of the aptamer-DNA duplex probe on the performance of displacement-based electrochemical aptamer sensors.

    Science.gov (United States)

    Pang, Jie; Zhang, Ziping; Jin, Haizhu

    2016-03-15

    Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors. Copyright

  13. Analysis of repetitive DNA in chromosomes by flow cytometry

    NARCIS (Netherlands)

    Brind'Amour, Julie; Lansdorp, Peter M.

    We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in

  14. DNA polymorphisms in the Sahiwal breed of Zebu cattle revealed by synthetic oligonucleotide probes

    International Nuclear Information System (INIS)

    Shashikanth; Yadav, B.R.

    2005-01-01

    Genomic DNA of 15 randomly selected unrelated animals and from two sire families (11 animals) of the Sahiwal breed of Zebu cattle were investigated. Four oligonucleotide probes - (GTG) 5 , (TCC) 5 , (GT) 8 and (GT) 12 - were used on genomic DNA digested with restriction enzymes AluI, HinfI, MboI, EcoRI and HaeIII in different combinations. All four probes produced multiloci fingerprints with differing levels of polymorphisms. Total bands and shared bands in the fingerprints of each individual were in the range of 2.5 to 23.0 KB. Band number ranged from 9 to 17, with 0.48 average band sharing. Probes (GT) 8 , (GT) 12 and (TCC) 5 produced fingerprinting patterns of medium to low polymorphism, whereas probe (GTG) 5 produced highly polymorphic patterns. Probe (GTG) 5 in combination with the HaeIII enzyme was highly polymorphic with a heterozygosity level of 0.85, followed by (GT) 8 , (TCC) 5 and (GT) 12 with heterozygosity levels of 0.70, 0.65 and 0.30, respectively. Probe GTG 5 or its complementary sequence CAC 5 produced highly polymorphic fingerprints, indicating that the probe can be used for analysing population structure, parentage verification and identifying loci controlling quantitative traits and fertility status. (author)

  15. DNA with Parallel Strand Orientation: A Nanometer Distance Study with Spin Labels in the Watson-Crick and the Reverse Watson-Crick Double Helix.

    Science.gov (United States)

    Wunnicke, Dorith; Ding, Ping; Yang, Haozhe; Seela, Frank; Steinhoff, Heinz-Jürgen

    2015-10-29

    Parallel-stranded (ps) DNA characterized by its sugar-phosphate backbones pointing in the same direction represents an alternative pairing system to antiparallel-stranded (aps) DNA with the potential to inhibit transcription and translation. 25-mer oligonucleotides were selected containing only dA·dT base pairs to compare spin-labeled nucleobase distances over a range of 10 or 15 base pairs in ps DNA with those in aps DNA. By means of the copper(I)-catalyzed Huisgen-Meldal-Sharpless alkyne-azide cycloaddition, the spin label 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl was clicked to 7-ethynyl-7-deaza-2'-deoxyadenosine or 5-ethynyl-2'-deoxyuridine to yield 25-mer oligonucleotides incorporating two spin labels. The interspin distances between spin labeled residues were determined by pulse EPR spectroscopy. The results reveal that in ps DNA these distances are between 5 and 10% longer than in aps DNA when the labeled DNA segment is located near the center of the double helix. The interspin distance in ps DNA becomes shorter compared with aps DNA when one of the spin labels occupies a position near the end of the double helix.

  16. The use of radionuclide DNA probe technology for epidemiological studies of tegumentary leishmaniasis in Mato Grosso state

    Directory of Open Access Journals (Sweden)

    Antero Silva Ribeiro de Andrade

    2005-10-01

    Full Text Available DNA hybridisation, using probes labelled with 32P, was used to type Leishmania samples isolated from patients living in endemic areas of Mato Grosso State (Brazil, and clinically diagnosed as having tegumentary leishmaniasis. kDNA cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes were used. The results showed that L. braziliensis is the predominant group infecting human patients in the state. Sixty-eight samples were typed, 64 samples (94.1% belonging to the L. braziliensis complex and only four (5.9% belonging to the L. mexicana complex. Accurate identification of the Leishmania permits better orientation of the medical follow-up, since clinical manifestations may vary depending on the complex to which the parasite belongs. The epidemiological information furnished by the identification of the Leishmania in given endemic area is also essential for the design of appropriate control measuresHibridização, utilizando sondas de DNA marcadas com 32P, foi utilizada para a tipagem de amostras de Leishmania isoladas de pacientes do estado do Mato Grosso (Brasil, diagnosticados clinicamente como portadores de leishmaniose tegumentar. Sondas de minicírculos clonados de kDNA, específicas para os complexos Leishmania mexicana e Leishmania braziliensis, foram utilizadas. Os resultados demonstraram que o complexo L. brasiliensis é o grupo predominante infectando pacientes humanos no estado do Mato Grosso. Foram tipadas 68 amostras: 64 (94,1% foram identificadas como pertencentes ao complexo L. brasiliensis e somente 4 (5,9% como pertencentes ao complexo L. mexicana. A tipagem de Leishmania é importante para um melhor acompanhamento médico, uma vez que as manifestações clínicas podem variar em função do complexo ao qual o parasita pertence. A informação fornecida pela identificação também é essencial para a definição das medidas de controle mais adequadas e compreensão da epidemiologia da

  17. A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

    International Nuclear Information System (INIS)

    Wang, Qingxiang; Ding, Yingtao; Gao, Feng; Jiang, Shulian; Zhang, Bin; Ni, Jiancong; Gao, Fei

    2013-01-01

    Graphical abstract: A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction between probe DNA and the sulfonic dye of 1-amino-2-naphthol-4-sulfonic acid that electrodeposited on a glassy carbon electrode. -- Highlights: •A versatile sulfonic dye of ANS was electrodeposited on a GCE. •A DNA biosensor was fabricated based on a facile sulfamide coupling reaction. •High probe DNA density of 3.18 × 10 13 strands cm −2 was determined. •A wide linear range and a low detection limit were obtained. -- Abstract: A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO 3 − ) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO 3 − layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO 3 − -AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO 3 − . The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH 3 ) 6 3+ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10 13 strands cm −2 and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen) 3 3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen) 3 3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10 −13 M to 1.0 × 10 −8 M with

  18. Application of DNA as a Smart Material

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther

    2011-01-01

    nanotechnology from the small assemblies in the beginning to the large and complex DNA structures of today. After the background chapter, the thesis consists of two parts. The first part comprises three projects regarding DNA origami (chapter 2–4). In the project described in chapter 2, DNA origami was exploited...... as an addressable platform for single molecule monitoring of chemical reactions. The addressability of the origami was crucial to the study, as it enabled the deduction of chemical identity of molecules from knowledge about position on the origami. Chapters 3 and 4 move into the third dimension, as they treat...... different aspects of 3D DNA origami. In the first project on 3D origami the folding process was investigated through incorporation of fluorophore-labelled staple strands. Facilitated by adaption of a technique for parallel enzymatic labelling of staple strands, the fate of multiple staple strands was probed...

  19. Identifying Fishes through DNA Barcodes and Microarrays.

    Directory of Open Access Journals (Sweden)

    Marc Kochzius

    2010-09-01

    Full Text Available International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S, cytochrome b (cyt b, and cytochrome oxidase subunit I (COI for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90% renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

  20. The use of radionuclide DNA probe technology in epidemiological studies of leishmaniasis

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical; Gomes, Rosangela Fatima; Melo, Maria Norma de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia]|[Cor Jesus Fontes Mato Grosso Univ., Cuiaba, MT (Brazil). Hospital Universitario Julio Muller

    2000-07-01

    Cutaneous and mucosal leishmaniasis are due to different species that belongs to Leishmania (Leishmania) mexicana complex and Leishmania (Viannia) braziliensis complex, respectively. Cutaneous leishmaniasis ulcers may persist for months to years but eventually they heal, while mucocutaneous leishmaniasis can result in destructive lesions on the nose, oral pharynx, lips or face. The specific diagnosis of the disease is important because of the high cost and toxicity of the treatment and the subsequent medical follow-up depends on the precise identification of the complex that causes the infection. The epidemiological information furnished by the identification of parasites of a given endemic region is also essential for the design of appropriate control measures. In this work we typed, using specific DNA probes labelled with {sup 32}P radionuclide, samples collected from patients living in endemic areas of Mato Grosso state. The results showed that L. braziliensis is the predominant group infecting human patients in the state. We have typed, up to the moment 68 samples. 64 samples (94.1%) belonged to the L. braziliensis complex and only 4 (5.9%) belonged to the L. mexicana complex. (author)

  1. Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Brandt, J.; Bojsen, K.

    1997-01-01

    genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both...... barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from...... transgenic tobacco was blocked by pyroglutamate, after the removal of which, N-terminal sequencing verified the transit signal-peptide cleavage site deduced from the cDNA sequence. Phenotype comparisons show that the constitutive expression of Prx8 lead to growth retardation. However, an infection assay...

  2. Detection of DNA fingerprints of cultivated rice by hybridization with a human minisatellite DNA probe

    International Nuclear Information System (INIS)

    Dallas, J.F.

    1988-01-01

    A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species

  3. Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    the molecule. In particular, surface plasmon resonance (SPR) sensors have been already demonstrated suitable for food-safety control, label-free screening for various disease markers in bodily fluids, as well as for real-time continuous monitoring of drug levels in intensive care environment. We envisage...... of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...

  4. Photoenzyme probes of photodamage to cells and cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B. M.

    1979-01-01

    Development of photoenzyme probes for detection of ultraviolet damage to cells and DNA is reviewed with special emphasis on a process using polyethylene glycol to induce cell fusion. Polyethylene glycol is easy to obtain and handle, is gentle to the cells and does not induce latent or productive virus infection; therefore, it may be a general method for insertion of exogenous enzymes into mammalian cells. (PCS)

  5. Peroxidases in nanostructures

    Directory of Open Access Journals (Sweden)

    Ana Maria eCarmona-Ribeiro

    2015-09-01

    Full Text Available Peroxidases are enzymes catalyzing redox reactions that cleave peroxides. Their active redox centers have heme, cysteine thiols, selenium, manganese and other chemical moieties. Peroxidases and their mimetic systems have several technological and biomedical applications such as environment protection, energy production, bioremediation, sensors and immunoassays design and drug delivery devices. The combination of peroxidases or systems with peroxidase-like activity with nanostructures such as nanoparticles, nanotubes, thin films, liposomes, micelles, nanoflowers, nanorods and others is often an efficient strategy to improve catalytic activity, targeting and reusability.

  6. Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

    International Nuclear Information System (INIS)

    Barfknecht, T.R.; Mecca, D.J.; Naismith, R.W.

    1988-01-01

    The results presented in this report demonstrate that an 18-20 hour exposure/ 3 H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin B1 and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18-20-hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18-20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10(-3) M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 X 10(-4) M when the 18-20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 X 10(-5) M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10(-5) M when the 18-20 hour exposure/labeling period was used

  7. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    Energy Technology Data Exchange (ETDEWEB)

    Serianni, A.S. [Univ. of Notre Dame, IN (United States)

    1994-12-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds.

  8. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    International Nuclear Information System (INIS)

    Serianni, A.S.

    1994-01-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds

  9. Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

    Directory of Open Access Journals (Sweden)

    Koji Hashimoto

    2016-05-01

    Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe. Keywords: Biosensor, DNA chip, Loop-mediated isothermal amplification (LAMP, Fluorescence detection, Gold substrate, Au/thiol bond

  10. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Science.gov (United States)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  11. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    International Nuclear Information System (INIS)

    Phuong Dinh Tam; Mai Anh Tuan; Tran Trung; Nguyen Duc Chien

    2009-01-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when emerged in double distilled water and kept refrigerated.

  12. Induction of DNA strand breaks in 14C-labelled cells

    International Nuclear Information System (INIS)

    Sundell-Bergman, S.; Johanson, K.J.

    1979-01-01

    Chinese hamster cells grown in vitro were labelled with 14 C-thymidine for 18 hours and after 3 hours in non-radioactive medium they were stored at 0 0 C for various periods ( 1 to 12 hours). During this treatment a number of DNA strand breaks were induced by 14 C decay which were not repaired at 0 0 C. The number of DNA strand breaks was determined using the DNA unwinding technique. At 0.5-1 dpm per cell a detectable number of DNA strand breaks were found. Treatment for six hours (1 dpm per cell) reduced the percentage of double-stranded DNA from 80 to 70%, corresponding to about 750 DNA strand breaks per cell. The rejoining of DNA strand breaks was studied after treatment for 12 hours at 0 0 C followed by incubation of the cells for various periods at 37 0 C. Most of the DNA strand breaks induced by 14 C decay at 0 0 C were repaired after incubation at 37 0 C for 15 minutes. Assuming an absorbed dose of 1.8 mGy per 14 C decay to the cell nucleus an RBE value close to 1 was found for internal irradiation from 14 C decay as compared with 60 Co-gamma irradiation. (author)

  13. New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

    Directory of Open Access Journals (Sweden)

    Hidetoshi Arakawa

    2012-04-01

    Full Text Available Horseradish peroxidase (HRP is generally used as a label enzyme in enzyme immunoassay (EIA. The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm.The measurable range of HRP was 1.0×10−18 to 1.0×10−15 mol/assay, with a detection limit of 1.0×10−18 mol/assay. The coefficient of variation (CV, n=8 was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH EIA using HRP as the label enzyme. Keywords: Sesamol, Fluorescence, Enzyme immunoassay (EIA, Horseradish peroxidase (HRP, Thyroid stimulating hormone (TSH

  14. Horseradish Peroxidase-Encapsulated Hollow Silica Nanospheres for Intracellular Sensing of Reactive Oxygen Species

    Science.gov (United States)

    Chen, Hsin-Yi; Wu, Si-Han; Chen, Chien-Tsu; Chen, Yi-Ping; Chang, Feng-Peng; Chien, Fan-Ching; Mou, Chung-Yuan

    2018-04-01

    Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

  15. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  16. DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

    Science.gov (United States)

    Balcioglu, Mustafa; Rana, Muhit; Robertson, Neil; Yigit, Mehmet V

    2014-08-13

    We controlled the fluorescence emission of a fluorescently labeled iron oxide nanoparticle using three different nanomaterials with ultraefficient quenching capabilities. The control over the fluorescence emission was investigated via spacing introduced by the surface-functionalized single-stranded DNA molecules. DNA molecules were conjugated on different templates, either on the surface of the fluorescently labeled iron oxide nanoparticles or gold and nanographene oxide. The efficiency of the quenching was determined and compared with various fluorescently labeled iron oxide nanoparticle and nanoquencher combinations using DNA molecules with three different lengths. We have found that the template for DNA conjugation plays significant role on quenching the fluorescence emission of the fluorescently labeled iron oxide nanoparticles. We have observed that the size of the DNA controls the quenching efficiency when conjugated only on the fluorescently labeled iron oxide nanoparticles by setting a spacer between the surfaces and resulting change in the hydrodynamic size. The quenching efficiency with 12mer, 23mer and 36mer oligonucleotides decreased to 56%, 54% and 53% with gold nanoparticles, 58%, 38% and 32% with nanographene oxide, 46%, 38% and 35% with MoS2, respectively. On the other hand, the presence, not the size, of the DNA molecules on the other surfaces quenched the fluorescence significantly with different degrees. To understand the effect of the mobility of the DNA molecules on the nanoparticle surface, DNA molecules were attached to the surface with two different approaches. Covalently immobilized oligonucleotides decreased the quenching efficiency of nanographene oxide and gold nanoparticles to ∼22% and ∼21%, respectively, whereas noncovalently adsorbed oligonucleotides decreased it to ∼25% and ∼55%, respectively. As a result, we have found that each nanoquencher has a powerful quenching capability against a fluorescent nanoparticle, which can be

  17. Microcantilver-based DNA hybridization sensors for Salmonella identification

    Directory of Open Access Journals (Sweden)

    Carlo Ricciardi

    2012-02-01

    Full Text Available The detection of pathogenic microorganisms in foods remains a challenging since the safety of foodstuffs has to be ensured by the food producing companies. Conventional methods for the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Biomolecular methods, are commonly used as a support for traditional techniques, thanks to their high sensitivity, specificity and not excessive costs. However, new methods like biosensors for example, can be an exciting alternative to the more traditional tecniques for the detection of pathogens in food. In this study we report Salmonella enterica serotype Enteritidis DNA detection through a novel class of label-free biosensors: microcantilevers (MCs. In general, MCs can operate as a microbalance and is used to detect the mass of the entities anchored to the cantilever surface using the decrease in the resonant frequency. We use DNA hybridization as model reaction system and for this reason, specific single stranded probe DNA of the pathogen and three different DNA targets (single-stranded complementary DNA, PCR product and serial dilutions of DNA extracted from S. Enteritidis strains were applied. Two protocols were reported in order to allow the probe immobilization on cantilever surface: i MC surface was functionalized with 3-aminopropyltriethoxysilane and glutaraldehyde and an amino-modified DNA probe was used; ii gold-coated sensors and thiolated DNA probes were used in order to generate a covalent bonding (Th-Au. For the first one, measures after hybridization with the PCR product showed related frequency shift 10 times higher than hybridization with complementary probe and detectable signals were obtained at the concentrations of 103 and 106 cfu/mL after hybridization with bacterial DNA. There are currently optimizations of the second protocol, where preliminary results have shown to be more uniform and therefore more precise within each of the

  18. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

    Science.gov (United States)

    Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

    2017-01-01

    Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

  19. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

    Directory of Open Access Journals (Sweden)

    Ahsan Munir

    2017-05-01

    Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

  20. Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

    Science.gov (United States)

    Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

    2008-08-22

    Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

  1. Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry. Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy

    DEFF Research Database (Denmark)

    Sigurdsson, Snorri; Vogel, Stefan; Shelke, Sandip

    2010-01-01

    and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair...... label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids...

  2. Blue shift of CdSe/ZnS nanocrystal-labels upon DNA-hybridization

    Directory of Open Access Journals (Sweden)

    Palme Klaus

    2008-05-01

    Full Text Available Abstract Luminescence color multiplexing is one of the most intriguing benefits, which might occur by using semiconductor Quantum Dots (QDs as labels for biomolecules. It was found, that the luminescence of QDs can be quenched, and replaced by a luminescence peak at approximately 460 nm on hybridization with certain regions of Arabidopsis thaliana tissue. This effect is site selective, and it is unclear whether it occurs due to an energy transfer process, or due to quenching and scattering of the excitation light. The article describes methods for phase-transfer of differently coloured, hydrophobically ligated QDs, coupling of DNA strands to the QD's surface, and hybridization of the labelled DNA to different cell types of Arabidopsis thaliana. The reason for the luminescence blue-shift was studied systematically, and narrowed down to the above mentioned causes.

  3. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Energy Technology Data Exchange (ETDEWEB)

    Phuong Dinh Tam; Mai Anh Tuan [International Training Institute for Materials Science (Viet Nam); Tran Trung [Department of Electrochemistry, Hung-Yen University of Technology and Education (Viet Nam); Nguyen Duc Chien [Institute of Engineering Physics, Hanoi University of Technology, 1 Dai Co Viet Road, Hanoi (Viet Nam)], E-mail: tr_trunghut@yahoo.com

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when emerged in double distilled water and kept refrigerated.

  4. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    International Nuclear Information System (INIS)

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-01-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts

  5. Methoxyphenol and Dihydrobenzofuran as Oxidizable Labels for Electrochemical Detection of DNA

    Czech Academy of Sciences Publication Activity Database

    Simonova, Anna; Balintová, Jana; Pohl, Radek; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2014-01-01

    Roč. 79, č. 12 (2014), s. 1703-1712 ISSN 2192-6506 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : DNA * electrochemistry * nucleotides * polymerase chain reactions * redox labeling Subject RIV: CC - Organic Chemistry Impact factor: 2.997, year: 2014

  6. Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

    Science.gov (United States)

    M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

    1994-01-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

  7. Cellular uptake of fluorophore-labeled glyco-DNA-gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Witten, Katrin G.; Ruff, Julie [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany); Mohr, Anne; Goertz, Dieter; Recker, Tobias; Rinis, Natalie [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Rech, Claudia; Elling, Lothar [RWTH Aachen University, Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering (Germany); Mueller-Newen, Gerhard [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Simon, Ulrich, E-mail: ulrich.simon@ac.rwth-aachen.de [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany)

    2013-10-15

    DNA-functionalized gold nanoparticles (AuNP-DNA) were hybridized with complementary di-N-acetyllactosamine-(di-LacNAc, [3Gal({beta}1-4)GlcNAc({beta}1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP-DNA-di-LacNAc. While AuNP-DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.

  8. [A new class of exciplex-formed probe detect of specific sequence DNA].

    Science.gov (United States)

    Li, Qing-Yong; Zu, Yuan-Gang; Lü, Hong-Yan; Wang, Li-Min

    2009-07-01

    The present research was to develop the exciplex-based fluorescence detection of DNA. A SNP-containing region of cytochrome P450 2C9 DNA systems was evaluated to define some of the structural and associated requirement of this new class of exciplex-formed probe, and a 24-base target was selected which contains single-nucleotide polymorphisms (SNP) in genes coding for cytochrome P450. The two probes were all 12-base to give coverage of a 24-base target region to ensure specificity within the human genome. Exciplex partners used in this study were prepared using analogous phosphoramide attachment to the 3'- or 5'-phosphate group of the appropriate oligonucleotide probes. The target effectively assembled its own detector by hybridization from components which were non-fluorescent at the detection wavelength, leading to the huge improvement in terms of decreased background. This research provides details of the effects of different partner, position of partners and different excitation wavelengths for the split-oligonucleotide probe system for exciplex-based fluorescence detection of DNA. This study demonstrates that the emission intensity of the excimer formed by new pyrene derivative is the highest in these excimer and exciplex, and the excimer is easy to be formed and not sensitive to the position of partners. However the exciplex formed by the new pyrene derivative and naphthalene emitted strongly at -505 nm with large Stokes shifts (120-130 nm), and the monomer emission at 390 and 410 nm is nearly zero. Excitation wavelength of 400 nm is the best for I(e505)/I(m410) (exciplex emission at 505 nm/monomer emission at 410 nm) of the exciplex. This method features lower background and high sensitivity. Moreover the exciplex is sensitive to the steric factor, different position of partners and microenvironment, so this exciplex system is promising and could be tried to identify the SNP genes.

  9. Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

    Science.gov (United States)

    Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

    2016-07-15

    A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Label-Free Ag+ Detection by Enhancing DNA Sensitized Tb3+ Luminescence

    Directory of Open Access Journals (Sweden)

    Kimberly Kleinke

    2016-08-01

    Full Text Available In this work, the effect of Ag+ on DNA sensitized Tb3+ luminescence was studied initially using the Ag+-specific RNA-cleaving DNAzyme, Ag10c. While we expected to observe luminescence quenching by Ag+, a significant enhancement was produced. Based on this observation, simple DNA oligonucleotide homopolymers were used with systematically varied sequence and length. We discovered that both poly-G and poly-T DNA have a significant emission enhancement by Ag+, while the absolute intensity is stronger with the poly-G DNA, indicating that a G-quadruplex DNA is not required for this enhancement. Using the optimized length of the G7 DNA (an oligo constituted with seven guanines, Ag+ was measured with a detection limit of 57.6 nM. The signaling kinetics, G7 DNA conformation, and the binding affinity of Tb3+ to the DNA in the presence or absence of Ag+ are also studied to reveal the mechanism of emission enhancement. This observation is useful not only for label-free detection of Ag+, but also interesting for the rational design of new biosensors using Tb3+ luminescence.

  11. Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

    Science.gov (United States)

    Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

    2014-06-17

    Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

  12. Preparation of goat and rabbit anti-camel immunoglobulin G whole molecule labeled with horseradish peroxidase

    Directory of Open Access Journals (Sweden)

    Eman Hussein Abdel-Rahman

    2017-01-01

    Full Text Available Aim: As the labeled anti-camel immunoglobulins (Igs with enzymes for enzyme-linked immunosorbent assay (ELISA are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP to save hard curacy. Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA. Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at −20°C as proved by ELISA. Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at −20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for

  13. Nucleobase modification as redox DNA labelling for electrochemical detection

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 40, č. 12 (2011), s. 5802-5814 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA AV ČR(CZ) IAA400040901; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : nucleotides * oligonucleotides * DNA * electrochemistry * redox labeling Subject RIV: CC - Organic Chemistry Impact factor: 28.760, year: 2011

  14. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  15. Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

    Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

  16. Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

    Science.gov (United States)

    Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

    2002-02-01

    We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

  17. Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

    Science.gov (United States)

    Douroupi, Triantafyllia G; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H

    2005-12-05

    A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.

  18. Guided mass spectrum labelling in atom probe tomography

    International Nuclear Information System (INIS)

    Haley, D.; Choi, P.; Raabe, D.

    2015-01-01

    Atom probe tomography (APT) is a valuable near-atomic scale imaging technique, which yields mass spectrographic data. Experimental correctness can often pivot on the identification of peaks within a dataset, this is a manual process where subjectivity and errors can arise. The limitations of manual procedures complicate APT experiments for the operator and furthermore are a barrier to technique standardisation. In this work we explore the capabilities of computer-guided ranging to aid identification and analysis of mass spectra. We propose a fully robust algorithm for enumeration of the possible identities of detected peak positions, which assists labelling. Furthermore, a simple ranking scheme is developed to allow for evaluation of the likelihood of each possible identity being the likely assignment from the enumerated set. We demonstrate a simple, yet complete work-chain that allows for the conversion of mass-spectra to fully identified APT spectra, with the goal of minimising identification errors, and the inter-operator variance within APT experiments. This work chain is compared to current procedures via experimental trials with different APT operators, to determine the relative effectiveness and precision of the two approaches. It is found that there is little loss of precision (and occasionally gain) when participants are given computer assistance. We find that in either case, inter-operator precision for ranging varies between 0 and 2 “significant figures” (2σ confidence in the first n digits of the reported value) when reporting compositions. Intra-operator precision is weakly tested and found to vary between 1 and 3 significant figures, depending upon species composition levels. Finally it is suggested that inconsistencies in inter-operator peak labelling may be the largest source of scatter when reporting composition data in APT. - Highlights: • Demonstration of a complete, but simple, automation chain for APT spectra analysis. • Algorithm for

  19. Guided mass spectrum labelling in atom probe tomography

    Energy Technology Data Exchange (ETDEWEB)

    Haley, D., E-mail: daniel.haley@materials.ox.ac.uk [Max-Planck-Institut für Eisenforschung, Max-Plack Straße 1, Düsseldorf (Germany); Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Choi, P.; Raabe, D. [Max-Planck-Institut für Eisenforschung, Max-Plack Straße 1, Düsseldorf (Germany)

    2015-12-15

    Atom probe tomography (APT) is a valuable near-atomic scale imaging technique, which yields mass spectrographic data. Experimental correctness can often pivot on the identification of peaks within a dataset, this is a manual process where subjectivity and errors can arise. The limitations of manual procedures complicate APT experiments for the operator and furthermore are a barrier to technique standardisation. In this work we explore the capabilities of computer-guided ranging to aid identification and analysis of mass spectra. We propose a fully robust algorithm for enumeration of the possible identities of detected peak positions, which assists labelling. Furthermore, a simple ranking scheme is developed to allow for evaluation of the likelihood of each possible identity being the likely assignment from the enumerated set. We demonstrate a simple, yet complete work-chain that allows for the conversion of mass-spectra to fully identified APT spectra, with the goal of minimising identification errors, and the inter-operator variance within APT experiments. This work chain is compared to current procedures via experimental trials with different APT operators, to determine the relative effectiveness and precision of the two approaches. It is found that there is little loss of precision (and occasionally gain) when participants are given computer assistance. We find that in either case, inter-operator precision for ranging varies between 0 and 2 “significant figures” (2σ confidence in the first n digits of the reported value) when reporting compositions. Intra-operator precision is weakly tested and found to vary between 1 and 3 significant figures, depending upon species composition levels. Finally it is suggested that inconsistencies in inter-operator peak labelling may be the largest source of scatter when reporting composition data in APT. - Highlights: • Demonstration of a complete, but simple, automation chain for APT spectra analysis. • Algorithm for

  20. Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1.

    Science.gov (United States)

    Sun, Linlin; Zhao, Qiang

    2018-03-01

    Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Direct immobilization and hybridization of DNA on group III nitride semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Xu Xiaobin; Jindal, Vibhu; Shahedipour-Sandvik, Fatemeh; Bergkvist, Magnus [College of Nanoscale Science and Engineering, University at Albany (SUNY), 255 Fuller Road, Albany, NY 12203 (United States); Cady, Nathaniel C. [College of Nanoscale Science and Engineering, University at Albany (SUNY), 255 Fuller Road, Albany, NY 12203 (United States)], E-mail: ncady@uamail.albany.edu

    2009-03-15

    A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.

  2. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Science.gov (United States)

    Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

    2014-11-01

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml-1 with a limit of detection of 0.16 ng ml-1.

  3. High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

    Science.gov (United States)

    Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

    2012-01-01

    Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

  4. Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, He, E-mail: mzhang_he@126.com; Fu, Xin; Hu, Jiayi; Zhu, Zhenjun

    2013-05-24

    Graphical abstract: A microfluidic beads-based nucleic acid sensor for sensitive detection of circulating tumor cells (CTCs) in the blood using multienzyme-nanoparticle amplification and quantum dots labels was developed. The chip-based CTCs analysis could detect reverse transcription-polymerase chain reaction (RT-PCR) products of tumor cell as low as 1 tumor cell (e.g. CEA expressing cell) in 1 mL blood sample. This microfluidic beads-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence. -- Highlights: •Combination of microfluidic bead-based platform and enzyme–probe–AuNPs is proposed. •The developed nucleic acid sensor could respond to 5 fM of tumor associated DNA. •Microfluidic platform and multienzyme-labeled AuNPs greatly enhanced sensitivity. •The developed nucleic acid sensor could respond to RT-PCR products of tumor cell as low as 1 tumor cell in 1 mL blood sample. •We report a sensitive nucleic acid sensor for detection of circulating tumor cells. -- Abstract: This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro

  5. Single DNA molecules as probes for interrogating silica surfaces after various chemical treatments

    International Nuclear Information System (INIS)

    Liu Xia; Wu Zhan; Nie Huagui; Liu Ziling; He Yan; Yeung, E.S.

    2007-01-01

    We examined the adsorption of single YOYO-1-labeled λ-DNA molecules at glass surfaces after treatment with various chemical cleaning methods by using total internal reflection fluorescence microscopy (TIRFM). The characteristics of these surfaces were further assessed using contact angle (CA) measurements and atomic force microscopy (AFM). By recording the real-time dynamic motion of DNA molecules at the liquid/solid interface, subtle differences in adsorption affinities were revealed. The results indicate that the driving force for adsorption of DNA molecules on glass surfaces is mainly hydrophobic interaction. We also found that surface topography plays a role in the adsorption dynamics

  6. Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

    Science.gov (United States)

    Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

    2006-09-01

    Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

  7. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  8. New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA.

    Science.gov (United States)

    Arakawa, Hidetoshi; Nakabayashi, Shigeo; Ohno, Ken-Ichi; Maeda, Masako

    2012-04-01

    Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10 -18 to 1.0×10 -15  mol/assay, with a detection limit of 1.0×10 -18  mol/assay. The coefficient of variation (CV, n =8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

  9. EPR spin probe and spin label studies of some low molecular and polymer micelles

    Science.gov (United States)

    Wasserman, A. M.; Kasaikin, V. A.; Timofeev, V. P.

    1998-12-01

    The rotational mobility of spin probes of different shape and size in low molecular and polymer micelles has been studied. Several probes having nitroxide fragment localized either in the vicinity of micelle interface or in the hydrocarbon core have been used. Upon increasing the number of carbon atoms in hydrocarbon chain of detergent from 7 to 13 (sodium alkyl sulfate micelles) or from 12 to 16 (alkyltrimethylammonium bromide micelles) the rotational mobility of spin probes is decreased by the factor 1.5-2.0. The spin probe rotational mobility in polymer micelles (the complexes of alkyltrimethylammonium bromides and polymethacrylic or polyacrylic acids) is less than mobility in free micelles of the same surfactants. The study of EPR-spectra of spin labeled polymethacrylic acid (PMA) indicated that formation of water soluble complexes of polymer and alkyltrimethylammonium bromides in alkaline solutions (pH 9) does not affect the polymer segmental mobility. On the other hand, the polymer complexes formation in slightly acidic water solution (pH 6) breaks down the compact PMA conformation, thus increasing the polymer segmental mobility. Possible structures of polymer micelles are discussed.

  10. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...

  11. Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant perioxidases

    DEFF Research Database (Denmark)

    Kjærsgård, I.V.H.; Jespersen, H.M.; Rasmussen, Søren Kjærsgård

    1997-01-01

    cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP la and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid...

  12. Synthesis of high specific activity tritium-labelled chloroethylcyclohexylnitrosourea and its application to the study of DNA modification

    International Nuclear Information System (INIS)

    Siew, E.L.; Habraken, Yvette; Ludlum, D.B.

    1991-01-01

    A small-scale synthesis of high specific activity, N-(2-chloro-2-[ 3 H-ethyl)-N'-cyclohexyl-N-nitrosourea ([ 3 H]-CCNU) has been accomplished from tritium-labelled ethanolamine. The product is pure by TLC and HPLC analysis and has been used successfully to modify DNA. The overall yield on radioactivity including losses in HPLC purification is approximately 4 percent. The availability of this tritium-labelled compound makes studies of DNA repair and of cellular resistance to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea possible. (author)

  13. Fetal sex determination in the first trimester of pregnancy using a Y chromosome-specific DNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Y.; Huang, S.; Chen, M.; Huang, Y.; Zhang, M.; Dong, J.; Ku, A.; Xu, S.

    1987-05-01

    Prenatal determination of fetal sex is important for the prevention of X-linked disorders such as hemophilia, Lesch-Nyhan syndrome and Duchenne muscular dystrophy. The complex procedures of prenatal diagnosis for X-linked disorders are unnecessary if the fetus is female, because usually no clinical symptoms ever appear in female. pY 3.4 probe used in this work for sex determination is a 3.4 kilobase human repeat sequence. The probe is specific for the Y chromosome of males and can be used for sex determination. The other prove pBLUR used in this paper as control is a widely dispersed, highly repeated human Alu family DNA sequence, represented equally in male and female DNA. On the basis of the relative densities of the autoradiographic spots produced by hybridization of fetal DNA with pY3.4 and pBLUR, the sex of fetus can be clearly identified. Further the authors can determine the radioactive intensity (cpm) of the hybridized DNA spots and the ratio of hybridization with Y3.4 to pBLUR (Y3.4/pBLUR x 10). Results show that the hybridization ratio of DNA from chorionic villi of male (1.03 +/- 0.24) is significantly higher than that of female (0.16 +/- 0.09). Therefore, sex determination of the fetus can be made, based on the ratio of pY3.4/pBLUR x 10. If necessary they can also use Southern hybridization with pY 3.4 probe of DNA isolated from chorionic villi to confirm the result of dot hybridization.

  14. An Internalin A Probe-Based Genosensor for Listeria monocytogenes Detection and Differentiation

    Directory of Open Access Journals (Sweden)

    Laura Bifulco

    2013-01-01

    Full Text Available Internalin A (InlA, a protein required for Listeria monocytogenes virulence, is encoded by the inlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect the inlA gene in Listeria strains without labelling the target DNA. The electrochemical sensor was obtained by immobilising an inlA gene probe (single-stranded oligonucleotide on the surfaces of screen-printed gold electrodes (Au-SPEs by means of a mercaptan-activated self-assembled monolayer (SAM. The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV using methylene blue (MB as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples of L. monocytogenes strains could be discriminated from other nonpathogenic Listeria species DNA with the inlA gene DNA probe genosensor.

  15. Seven novel probe systems for real-time PCR provide absolute single-base discrimination, higher signaling, and generic components.

    Science.gov (United States)

    Murray, James L; Hu, Peixu; Shafer, David A

    2014-11-01

    We have developed novel probe systems for real-time PCR that provide higher specificity, greater sensitivity, and lower cost relative to dual-labeled probes. The seven DNA Detection Switch (DDS)-probe systems reported here employ two interacting polynucleotide components: a fluorescently labeled probe and a quencher antiprobe. High-fidelity detection is achieved with three DDS designs: two internal probes (internal DDS and Flip probes) and a primer probe (ZIPR probe), wherein each probe is combined with a carefully engineered, slightly mismatched, error-checking antiprobe. The antiprobe blocks off-target detection over a wide range of temperatures and facilitates multiplexing. Other designs (Universal probe, Half-Universal probe, and MacMan probe) use generic components that enable low-cost detection. Finally, single-molecule G-Force probes employ guanine-mediated fluorescent quenching by forming a hairpin between adjacent C-rich and G-rich sequences. Examples provided show how these probe technologies discriminate drug-resistant Mycobacterium tuberculosis mutants, Escherichia coli O157:H7, oncogenic EGFR deletion mutations, hepatitis B virus, influenza A/B strains, and single-nucleotide polymorphisms in the human VKORC1 gene. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  16. Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

    KAUST Repository

    Wang, Zhijuan

    2011-05-01

    Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

  17. Scanning Probe Optical Tweezers: a new tool to study DNA-protein interactions

    NARCIS (Netherlands)

    Huisstede, J.H.G.

    2006-01-01

    The main goal of the work described in this thesis is to construct a microscope in which OT and scanning probe microscopy (SPM) are combined, to be able to localize proteins while simultaneously controlling the tension within the DNA molecule. This apparatus enables the study of the effect of

  18. Construction of recombinant DNA clone for bovine viral diarrhea virus

    International Nuclear Information System (INIS)

    Yeo, S.G.; Cho, H.J.; Masri, S.A.

    1992-01-01

    Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

  19. Chemiprobe, a nonradioactive system for labeling nucleic acid. Principles and applications.

    Science.gov (United States)

    Nur, I; Reinhartz, A; Hyman, H C; Razin, S; Herzberg, M

    1989-01-01

    The Chemiprobe Kit provides a complete system for nonradioactive labeling of DNA probes and their detection in hybridization studies. The system is highly sensitive, permitting the detection of 0.2-0.4 pg DNA which allows detection of a single gene sequence in 0.5-1 microgram of bacterial DNA or in 3-5 micrograms of mammalian DNA. In this paper the authors show that the rRNA genes of M. capricolum can be detected by using only 50 ng/ml of sulfonated probe cloned from another mycoplasma, M. pneumoniae. The Chemiprobe system has been successfully used in the detection of the single copy human gene for glucocerobrosidase from total embryonic DNA by hybridization to a specific sulfonated cDNA. 5 x 10(4) M. pneumoniae cells can be detected either free or mixed with sputum using a standard dot blot technique: mycoplasma cells were lysed by a mucolytic agent, denaturated by NaOH, immobilized on a nylon membrane filter, and then hybridized with pPN4, a plasmid DNA probe specific for M. pneumoniae. The resulting hybrids were then detected by the standard Chemiprobe procedure. A new kit based on the Chemiprobe system has been designed especially for the detection of mycoplasmas in tissue culture. This kit has been tested on 70 random samples collected from tissue culture fluids from 11 different sources. Of these, 42 were found to be contaminated by the Chemiprobe procedure, whereas 41 were found to be contaminated by classical microbiological methods. No false negatives were found.

  20. Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

    Science.gov (United States)

    Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

    2016-04-15

    In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  2. Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Haiying [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China); Department of Chemistry, Yuncheng University, Yuncheng 044300 (China); Li, Zhejian; Shan, Meng; Li, Congcong; Qi, Honglan; Gao, Qiang [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China); Wang, Jinyi [College of Science and College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Zhang, Chengxiao, E-mail: cxzhang@snnu.edu.cn [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China)

    2015-03-10

    Highlights: • A novel biosensor was developed for the detection of prostate cancer cells. • The selectivity of the biosensor was improved using antibody as capture probe. • The biosensor showed the low extremely detection limit of 2.6 × 10{sup 2} cells mL{sup −1}. • The ruthenium complex-labelled WGA can be transported in the cell vesicles. - Abstract: A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10{sup 2} to 3.0 × 10{sup 4} cells mL{sup −1}, with a detection limit of 2.6 × 10{sup 2} cells mL{sup −1}. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL{sup −1}. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.

  3. Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

    International Nuclear Information System (INIS)

    Yang, Haiying; Li, Zhejian; Shan, Meng; Li, Congcong; Qi, Honglan; Gao, Qiang; Wang, Jinyi; Zhang, Chengxiao

    2015-01-01

    Highlights: • A novel biosensor was developed for the detection of prostate cancer cells. • The selectivity of the biosensor was improved using antibody as capture probe. • The biosensor showed the low extremely detection limit of 2.6 × 10 2 cells mL −1 . • The ruthenium complex-labelled WGA can be transported in the cell vesicles. - Abstract: A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10 2 to 3.0 × 10 4 cells mL −1 , with a detection limit of 2.6 × 10 2 cells mL −1 . The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL −1 . The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes

  4. Synthesis of high specific activity tritium-labelled chloroethylcyclohexylnitrosourea and its application to the study of DNA modification

    Energy Technology Data Exchange (ETDEWEB)

    Siew, E.L. (State Univ. of New York, Albany, NY (USA). Dept. of Chemistry); Habraken, Yvette; Ludlum, D.B. (Massachusetts Univ., Worcester, MA (USA). Medical School)

    1991-02-01

    A small-scale synthesis of high specific activity, N-(2-chloro-2-{sup 3}H-ethyl)-N'-cyclohexyl-N-nitrosourea ({sup 3}H-CCNU) has been accomplished from tritium-labelled ethanolamine. The product is pure by TLC and HPLC analysis and has been used successfully to modify DNA. The overall yield on radioactivity including losses in HPLC purification is approximately 4 percent. The availability of this tritium-labelled compound makes studies of DNA repair and of cellular resistance to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea possible. (author).

  5. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  6. Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

    Science.gov (United States)

    Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

    2001-03-01

    The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

  7. In situ detection of tandem DNA repeat length

    Energy Technology Data Exchange (ETDEWEB)

    Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

    1996-11-01

    A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

  8. Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

    Science.gov (United States)

    Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Chen, Yin; Neufeld, Josh D; Murrell, J Colin

    2016-01-01

    Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

  9. Physical mapping of a 330 X 10(3)-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination

    International Nuclear Information System (INIS)

    Komano, T.; Inouye, S.; Inouye, M.

    1985-01-01

    Myxococcus xanthus was pulse-labeled with [ 3 H]thymidine immediately after germination of dimethyl sulfoxide-induced spores. The restriction enzyme digests of the total chromosomal DNA from the pulse- labeled cells were analyzed by one-dimensional as well as two- dimensional agarose gel electrophoresis. Four PstI fragments preferentially labeled at a very early stage of germination were cloned into the unique PstI site of pBR322. By using these clones as probes, a restriction enzyme map was established covering approximately 6% of the total M. xanthus genome (330 X 10(3) base pairs). The distribution of the specific activities of the restriction fragments pulse-labeled after germination suggests a bidirectional mode of DNA replication from a fixed origin

  10. The DNA hybridization assay using single-walled carbon nanotubes as ultrasensitive, long-term optical labels

    International Nuclear Information System (INIS)

    Hwang, Eung-Soo; Cao, Chengfan; Hong, Sanghyun; Jung, Hye-Jin; Cha, Chang-Yong; Choi, Jae-Boong; Kim, Young-Jin; Baik, Seunghyun

    2006-01-01

    Single walled carbon nanotubes (SWNTs) exhibit strong Raman signals as well as fluorescence emissions in the near infrared region. Such signals do not blink or photobleach under prolonged excitation, which is an advantage in optical nano-biomarker applications. In this paper, we present single-stranded DNA conjugated SWNT probes to locate a particular sequence of DNA within a complex genome. Chromosomal DNAs of human fibroblasts and Escherichia coli are used as a target and a control, respectively. Southern blotting, which uses photostable Raman signals of nanotubes instead of fluorescent dyes, demonstrates excellent sensitivity and specificity of the probes. The results show that SWNTs may be used as generic nano-biomarkers for the precise detection of specific kinds of genes

  11. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  12. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  13. DNA-based stable isotope probing: a link between community structure and function

    Czech Academy of Sciences Publication Activity Database

    Uhlík, Ondřej; Ječná, K.; Leigh, M. B.; Macková, Martina; Macek, Tomáš

    2009-01-01

    Roč. 407, č. 12 (2009), s. 3611-3619 ISSN 0048-9697 Grant - others:GA MŠk(CZ) 2B08031 Program:2B Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA-based stable isotope probing * microbial diversity * bioremediation Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.905, year: 2009

  14. Luminescent platinum(II) complexes with functionalized N-heterocyclic carbene or diphosphine selectively probe mismatched and abasic DNA

    OpenAIRE

    Che, CM; Chen, T; To, WP; Zou, T; FUNG, SK; Lok, CN; YANG, C; Cao, B

    2016-01-01

    The selective targeting of mismatched DNA overexpressed in cancer cells is an appealing strategy in designing cancer diagnosis and therapy protocols. Few luminescent probes that specifically detect intracellular mismatched DNA have been reported. Here we used Pt(II) complexes with luminescence sensitive to subtle changes in the local environment and report several Pt(II) complexes that selectively bind to and identify DNA mismatches. We evaluated the complexes' DNA-binding characteristics by ...

  15. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  16. Electrokinetic acceleration of DNA hybridization in microsystems.

    Science.gov (United States)

    Lei, Kin Fong; Wang, Yun-Hsiang; Chen, Huai-Yi; Sun, Jia-Hong; Cheng, Ji-Yen

    2015-06-01

    In this work, electrokinetic acceleration of DNA hybridization was investigated by different combinations of frequencies and amplitudes of actuating electric signals. Because the frequencies from low to high can induce different kinds of electrokinetic forces, i.e., electroosmotic to electrothermal forces, this work provides an in-depth investigation of electrokinetic enhanced hybridization. Concentric circular Cr/Au microelectrodes of 350 µm in diameter were fabricated on a glass substrate and probe DNA was immobilized on the electrode surface. Target DNA labeled with fluorescent dyes suspending in solution was then applied to the electrode. Different electrokinetic forces were induced by the application of different electric signals to the circular microelectrodes. Local microfluidic vortexes were generated to increase the collision efficiency between the target DNA suspending in solution and probe DNA immobilized on the electrode surface. DNA hybridization on the electrode surface could be accelerated by the electrokinetic forces. The level of hybridization was represented by the fluorescent signal intensity ratio. Results revealed that such 5-min dynamic hybridization increased 4.5 fold of signal intensity ratio as compared to a 1-h static hybridization. Moreover, dynamic hybridization was found to have better differentiation ability between specific and non-specific target DNA. This study provides a strategy to accelerate DNA hybridization in microsystems. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    Science.gov (United States)

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

  18. Development and Validation of A Spectrofluorimetric Determination of Calf Thymus DNA Using a Terbium-Danofloxacin Probe

    Directory of Open Access Journals (Sweden)

    Naser Soltani

    2016-03-01

    Full Text Available Background: Analysis of biomolecules is required in many biomedical research areas. A spectrofluorimetric method is proposed for determination of calf thymus DNA (ctDNA based on the fluorescence enhancement of terbium-danofloxacin (Tb3+-Dano in the presence of ctDNA. Methods: A probe with maximum excitation and emission wavelengths of 347 nm and 545 nm, respectively, was developed. The enhanced fluorescence intensity of Tb3+-Dano system was proportional to the concentration of ctDNA. The effective factors and the optimum conditions for the determination of ctDNA were studied. Under the optimum conditions of [Tris buffer]= 0.01 mol L-1 (pH 7.8, [ Tb3+]= 1×10-5 mol L-1 and [Dano]= 5×10-5 mol L-1, the maximum response was achieved. The developed method was evaluated in terms of accuracy, precision and limit of detection. Results: The linear concentration range for quantification of ctDNA was 36-3289 ng mL-1 and the detection limit (S/N=3 was 8 ng mL-1. The concentration of DNA extracted from Escherichia coli as an extracted sample was also determined using the developed probe. The concentration of DNA in extracted sample was determined using UV assay and developed method, the results were satisfactory. Conclusion: The proposed method is a simple, practical and relatively interference free method to follow up the concentrations of ctDNA.

  19. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    International Nuclear Information System (INIS)

    Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

    2014-01-01

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml −1 with a limit of detection of 0.16 ng ml −1

  20. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Swati; Kumar, Ashok, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007 (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India); Khare, Shashi [National Centre for Disease Control, Sham Nath Marg, Delhi 110054 (India); Mulchandani, Ashok [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States); Rajesh, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-National Physical Laboratory, Dr. K. S. Krishnan Road, New Delhi 110012 (India)

    2014-11-24

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml{sup −1} with a limit of detection of 0.16 ng ml{sup −1}.

  1. Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme

    Science.gov (United States)

    Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

    2018-06-01

    Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562 nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50 ng mL-1 with the limit detection of 9.899 ng mL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108 CFU mL-1 in real samples with a detection limit of 320 CFU mL-1.

  2. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    Science.gov (United States)

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-08-10

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

  3. Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

    Science.gov (United States)

    Carmichael, P L; Sardar, S; Crooks, N; Neven, P; Van Hoof, I; Ugwumadu, A; Bourne, T; Tomas, E; Hellberg, P; Hewer, A J; Phillips, D H

    1999-02-01

    Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

  4. Detection of DNA hybridization based on SnO2 nanomaterial enhanced fluorescence

    International Nuclear Information System (INIS)

    Gu Cuiping; Huang Jiarui; Ni Ning; Li Minqiang; Liu Jinhuai

    2008-01-01

    In this paper, enhanced fluorescence emissions were firstly investigated based on SnO 2 nanomaterial, and its application in the detection of DNA hybridization was also demonstrated. The microarray of SnO 2 nanomaterial was fabricated by the vapour phase transport method catalyzed by patterned Au nanoparticles on a silicon substrate. A probe DNA was immobilized on the substrate with patterned SnO 2 nanomaterial, respectively, by covalent and non-covalent linking schemes. When a fluorophore labelled target DNA was hybridized with a probe DNA on the substrate, fluorescence emissions were only observed on the surface of SnO 2 nanomaterial, which indicated the property of enhancing fluorescence signals from the SnO 2 nanomaterial. By comparing the different fluorescence images from covalent and non-covalent linking schemes, the covalent method was confirmed to be more effective for immobilizing a probe DNA. With the combined use of SnO 2 nanomaterial and the covalent linking scheme, the target DNA could be detected at a very low concentration of 10 fM. And the stability of SnO 2 nanomaterial under the experimental conditions was also compared with silicon nanowires. The findings strongly suggested that SnO 2 nanomaterial could be extensively applied in detections of biological samples with enhancing fluorescence property and high stability

  5. A peroxidase gene expressed during early developmental stages of the parasitic plant Orobanche ramosa.

    Science.gov (United States)

    González-Verdejo, Clara Isabel; Barandiaran, Xabier; Moreno, Maria Teresa; Cubero, José Ignacio; Di Pietro, Antonio

    2006-01-01

    Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control the early stages of host infection in Orobanche are poorly understood. In the present study, the role of peroxidase has been examined during pre-infection growth and development of O. ramosa, using an in vitro model system. Peroxidase activity was histochemically localized at the tips of actively growing radicles and nascent attachment organs. Addition of exogenous catalase resulted in a significant reduction in the apical growth rate of the radicle. The prx1 gene encoding a putative class III peroxidase was cloned from a cDNA library of O. ramosa and was found to be expressed specifically during the early stages of the parasitic life cycle. The exogenous addition of sucrose resulted in significantly reduced prx1 transcript levels and in a dramatic change in radicle development from polarized apical growth to isotropic growth and the formation of tubercle-like structures. The results indicate an important role of peroxidases during the early parasitic stages of Orobanche.

  6. Spiky gold shells on magnetic particles for DNA biosensors.

    Science.gov (United States)

    Bedford, Erin E; Boujday, Souhir; Pradier, Claire-Marie; Gu, Frank X

    2018-05-15

    Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Development of a dot blot assay using gene probes for the detection of enteroviruses in water

    International Nuclear Information System (INIS)

    Margolin, A.B.

    1986-01-01

    Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with 32 P dCTP and 32 P dATP to a specific activity greater then 1.0 x 10 9 cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses

  8. Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

    OpenAIRE

    Chaves, F; Yang, Z; el Hajj, H; Alonso, M; Burman, W J; Eisenach, K D; Dronda, F; Bates, J H; Cave, M D

    1996-01-01

    A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more cop...

  9. Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

    Science.gov (United States)

    Wood, S; Starr, T V; Shukin, R J

    1986-01-01

    We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

  10. "Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E

    2011-03-01

    A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

  11. Machine Learned Replacement of N-Labels for Basecalled Sequences in DNA Barcoding.

    Science.gov (United States)

    Ma, Eddie Y T; Ratnasingham, Sujeevan; Kremer, Stefan C

    2018-01-01

    This study presents a machine learning method that increases the number of identified bases in Sanger Sequencing. The system post-processes a KB basecalled chromatogram. It selects a recoverable subset of N-labels in the KB-called chromatogram to replace with basecalls (A,C,G,T). An N-label correction is defined given an additional read of the same sequence, and a human finished sequence. Corrections are added to the dataset when an alignment determines the additional read and human agree on the identity of the N-label. KB must also rate the replacement with quality value of in the additional read. Corrections are only available during system training. Developing the system, nearly 850,000 N-labels are obtained from Barcode of Life Datasystems, the premier database of genetic markers called DNA Barcodes. Increasing the number of correct bases improves reference sequence reliability, increases sequence identification accuracy, and assures analysis correctness. Keeping with barcoding standards, our system maintains an error rate of percent. Our system only applies corrections when it estimates low rate of error. Tested on this data, our automation selects and recovers: 79 percent of N-labels from COI (animal barcode); 80 percent from matK and rbcL (plant barcodes); and 58 percent from non-protein-coding sequences (across eukaryotes).

  12. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  13. Accurate quantification of supercoiled DNA by digital PCR

    Science.gov (United States)

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  14. Competitive photometric enzyme immunoassay for fullerene C60 and its derivatives using a fullerene conjugated to horseradish peroxidase

    International Nuclear Information System (INIS)

    Hendrickson, Olga D.; Smirnova, Natalya I.; Zherdev, Anatoly V.; Dzantiev, Boris B.; Sveshnikov, Peter G.

    2016-01-01

    The article describes a highly sensitive single-step microplate enzyme immunoassay of the ELISA type for fullerene C 60 and its derivatives. Monoclonal anti-fullerene antibodies and a conjugate between fullerene and horseradish peroxidase were used as specific reagents. A direct competitive ELISA was carried out that was based on antibodies immobilized in the well of a microtiter plate, a peroxidase-labeled antigen, and detection via the dye formed from 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Both pristine fullerene C 60 and its water-soluble forms can be determined. The detection limits are 1.5 ng∙mL −1 for fullerene C 60 , and between 0.1 and 1.3 ng∙mL −1 for its derivatives. This ELISA format allows for almost two-fold reduction of the time needed for the assay in comparison to indirect scheme with labeled antibodies. (author)

  15. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogona...

  16. Design of photoaffinity labeling probes derived from 3,4,5-trimethylfuran-2(5H)-one for mode of action elucidation

    Czech Academy of Sciences Publication Activity Database

    Pošta, Martin; Soós, V.; Beier, Petr

    2016-01-01

    Roč. 72, 27/28 (2016), s. 3809-3817 ISSN 0040-4020 Institutional support: RVO:61388963 Keywords : photoaffinity labeling probes * protein cross-linking * smoke * butenolides Subject RIV: CC - Organic Chemistry Impact factor: 2.651, year: 2016

  17. Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer

    International Nuclear Information System (INIS)

    Xia, Peng; An, Han-Xiang; Dang, Cheng-Xue; Radpour, Ramin; Kohler, Corina; Fokas, Emmanouil; Engenhart-Cabillic, Rita; Holzgreve, Wolfgang; Zhong, Xiao Yan

    2009-01-01

    Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters. Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein. The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed. Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer

  18. Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

    Directory of Open Access Journals (Sweden)

    R.C. Silva

    1998-09-01

    Full Text Available Adrenocortical autoantibodies (ACA, present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13 and granulomatous (N = 9. ACA-PLPA were detected in 10/22 (45% patients: 9/13 (69% with the idiopathic form and 1/9 (11% with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%: 10/13 (77% with the idiopathic form and 1/9 (11% with the granulomatous form. Twelve of the 13 idiopathic addisonians (92% were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time. However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.

  19. Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

    Science.gov (United States)

    Qian, Yong; Wang, Chunyan; Gao, Fenglei

    2015-01-15

    A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Ultra-small particles of iron oxide as peroxidase for immunohistochemical detection

    International Nuclear Information System (INIS)

    Wu Yihang; Song Mengjie; Zhang Xiaoqing; Zhang Yu; Wang Chunyu; Gu Ning; Xin Zhuang; Li Suyi

    2011-01-01

    Dimercaptosuccinic acid (DMSA) modified ultra-small particles of iron oxide (USPIO) were synthesized through a two-step process. The first step: oleic acid (OA) capped Fe 3 O 4 (OA-USPIO) were synthesized by a novel oxidation coprecipitation method in H 2 O/DMSO mixing system, where DMSO acts as an oxidant simultaneously. The second step: OA was replaced by DMSA to obtain water-soluble nanoparticles. The as-synthesized nanoparticles were characterized by TEM, FTIR, TGA, VSM, DLS, EDS and UV-vis. Hydrodynamic sizes and Peroxidase-like catalytic activity of the nanoparticles were investigated. The hydrodynamic sizes of the nanoparticles (around 24.4 nm) were well suited to developing stable nanoprobes for bio-detection. The kinetic studies were performed to quantitatively evaluate the catalytic ability of the peroxidase-like nanoparticles. The calculated kinetic parameters indicated that the DMSA-USPIO possesses high catalytic activity. Based on the high activity, immunohistochemical experiments were established: using low-cost nanoparticles as the enzyme instead of expensive HRP, Nimotuzumab was conjugated onto the surface of the nanoparticles to construct a kind of ultra-small nanoprobe which was employed to detect epidermal growth factor receptor (EGFR) over-expressed on the membrane of esophageal cancer cell. The proper sizes of the probes and the result of membranous immunohistochemical staining suggest that the probes can be served as a useful diagnostic reagent for bio-detection.

  1. Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes.

    Science.gov (United States)

    Small, A L; McFall-Ngai, M J

    1999-03-15

    An enzyme with similarities to myeloperoxidase, the antimicrobial halide peroxidase in mammalian neutrophils, occurs abundantly in the light organ tissue of Euprymna scolopes, a squid that maintains a beneficial association with the luminous bacterium Vibrio fischeri. Using three independent assays typically applied to the analysis of halide peroxidase enzymes, we directly compared the activity of the squid enzyme with that of human myeloperoxidase. One of these methods, the diethanolamine assay, confirmed that the squid peroxidase requires halide ions for its activity. The identification of a halide peroxidase in a cooperative bacterial association suggested that this type of enzyme can function not only to control pathogens, but also to modulate the interactions of host animals with their beneficial partners. To determine whether the squid peroxidase functions under both circumstances, we examined its distribution in a variety of host tissues, including those that typically interact with bacteria and those that do not. Tissues interacting with bacteria included those that have specific cooperative associations with bacteria (i.e., the light organ and accessory nidamental gland) and those that have transient nonspecific interactions with bacteria (i.e., the gills, which clear the cephalopod circulatory system of invading microorganisms). These bacteria-associated tissues were compared with the eye, digestive gland, white body, and ink-producing tissues, which do not typically interact directly with bacteria. Peroxidase enzyme assays, immunocytochemical localization, and DNA-RNA hybridizations showed that the halide-dependent peroxidase is consistently expressed in high concentration in tissues that interact bacteria. Elevated levels of the peroxidase were also found in the ink-producing tissues, which are known to have enzymatic pathways associated with antimicrobial activity. Taken together, these data suggest that the host uses a common biochemical response to

  2. The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

    Science.gov (United States)

    Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

    2014-01-01

    Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

  3. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...

  4. Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes

    International Nuclear Information System (INIS)

    Hyypiae, T.

    1985-01-01

    The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an enzyme immunoassay for adenovirus hexon antigen which was used as a reference test. Sixteen specimens positive by the enzyme immunoassay also were positive in the two nucleic acid hybridization tests, and the remaining eight specimens were negative in all of the tests. The results indicate that nucleid acid hybridization tests with both radioactive and nonradioactive probes can be used for diagnosis of microbial infections

  5. DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

    Science.gov (United States)

    Bronder, Thomas S; Poghossian, Arshak; Scheja, Sabrina; Wu, Chunsheng; Keusgen, Michael; Mewes, Dieter; Schöning, Michael J

    2015-09-16

    Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

  6. Synthesis of novel fluorescent probe Tb(III)-7-carboxymethoxy-4-methylcoumarin complex for sensing of DNA

    International Nuclear Information System (INIS)

    Hussein, Belal H.M.; Azab, Hassan A.; Fathalla, Walid; Ali, Sherin A.M.

    2013-01-01

    New fluorescent probe Tb(III) (7-carboxymethoxy-4-methylcoumarin)2(SCN) (C2H5OH)(H2O) was synthesized and characterized by spectroscopy and thermal analysis. The absorption and fluorescence spectra of 7-carboxymethoxy-4-methylcoumarin (CMMC) and Tb(III)–CMMC complex have been measured in different solvents. The interactions of Tb(III)–CMMC complex with calf thymus nucleic acid (CT-DNA) have been investigated using steady state fluorescence measurements. The changes in the fluorescence intensity have been used for the quantitative determination of DNA with LOD of 3.45 ng in methanol–water (9:1, v/v). The association constants of DNA with Tb(III)–CMMC complex was found to be 2.62×1010 M −1 . - Highlights: ► New fluorescent probe Terbium (III)-7-carboxy methoxy-4-methylcoumarin complex has been synthesized and characterized. ► FTIR spectrum of Tb(III)-complex shows a characteristic band for thiocyanate group. ► DNA interaction with Terbium (III)-7-carboxy methoxy-4-methylcoumarin has been studied by fluorescence techniques. ► The change in the fluorescence intensity has been used for the quantitative determination of DNA. ► The result was better than most of the well-known methods including the ethidium bromide method.

  7. Wiring of heme enzymes by methylene-blue labeled dendrimers

    DEFF Research Database (Denmark)

    Álvarez-Martos, Isabel; Shahdost-fard, Faezeh; Ferapontova, Elena

    2017-01-01

    Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme- and moli......Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme......- and molibdopterin-containing sulfite oxidase (SOx), wired to gold by the methylene blue (MB)-labeled polyamidoamine (PAMAM) dendrimers. The enzymes’ electrochemical transformation and bioelectrocatalytic function could be followed at both unlabeled and MB-labeled dendrimer-modified electrodes with the formal redox......, optimization of bioelectrocatalysis of complex intermembrane and, possibly, membrane enzymes....

  8. One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    International Nuclear Information System (INIS)

    Liu, Ziping; Su, Xingguang

    2016-01-01

    Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

  9. Near-infrared dyes and upconverting phosphors as biomolecule labels and probes

    Science.gov (United States)

    Patonay, Gabor; Strekowski, Lucjan; Nguyen, Diem-Ngoc; Seok, Kim Jun

    2007-02-01

    Near-Infrared (NIR) absorbing chromophores have been used in analytical and bioanalytical chemistry extensively, including for determination of properties of biomolecules, DNA sequencing, immunoassays, capillary electrophoresis (CE) separations, etc. The major analytical advantages of these dyes are low background interference and high molar absorptivities. NIR dyes have additional advantages due to their sensitivity to microenvironmental changes. Spectral changes induced by the microenvironment are not desirable if the labels are used as a simple reporting group, e.g., during a biorecognition reaction. For these applications upconverting phosphors seem to be a better choice. There are several difficulties in utilizing upconverting phosphors as reporting labels. These are: large physical size, no reactive groups and insolubility in aqueous systems. This presentation will discuss how these difficulties can be overcome for bioanalytical and forensic applications. During these studies we also have investigated how to reduce physical size of the phosphor by simple grinding without losing activity and how to attach reactive moiety to the phosphor to covalently bind to the biomolecule of interest. It has to be emphasized that the described approach is not suitable for medical applications and the results of this research are not applicable in medical applications. For bioanalytical and forensic applications upconverting phosphors used as reporting labels have several advantages. They are excited with lasers that are red shifted respective to phosphorescence, resulting in no light scatter issues during detection. Also some phosphors are excited using eye safe lasers. In addition energy transfer to NIR dyes is possible, allowing detection schemes using donor-acceptor pairs. Data is presented to illustrate the feasibility of this phenomenon. If microenvironmental sensitivity is required, then specially designed NIR dyes can be used as acceptor labels. Several novel dyes

  10. Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

    2006-01-01

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

  11. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    Science.gov (United States)

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. DNA hybridization sensor based on pentacene thin film transistor.

    Science.gov (United States)

    Kim, Jung-Min; Jha, Sandeep Kumar; Chand, Rohit; Lee, Dong-Hoon; Kim, Yong-Sang

    2011-01-15

    A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  14. Biofunctionalization of ZnO nanowires for DNA sensory applications

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, Ulrich Christian; Gnauck, Martin; Ronning, Carsten [Institute of Solid State Physics, University of Jena, Max-Wien-Platz 1, D-07743 Jena (Germany); Moeller, Robert; Rudolph, Bettina; Fritzsche, Wolfgang [Institut fuer Photonische Technologien e.V., Albert-Einstein-Strasse 9, D-07745 Jena (Germany)

    2011-07-01

    In recent years, DNA detecting systems have received a growing interest due to promising fields of application like DNA diagnostics, gene analysis, virus detection or forensic applications. Nanowire-based DNA biosensor allows both miniaturization and easy continuous monitoring of a detection signal by electrical means. The label free detection scheme based on electrochemical changes of the surface potential during immobilization of specific DNA probes was heretofore mainly studied for silicon. In this work a surface decoration process with bifunctional molecules known as silanization was applied to VLS-grown ZnO nanowires which both feature a large sensitivity for surface modification, are biocompatible and easy to synthesize as well. Successfully bound DNA was proved by fluorescence microscopy. Dielectrophoresis (DEP) was chosen and optimized for quickly contacting the ZnO nanowires. Furthermore, electrical signal characterization was performed in preparation for DNA sensory applications.

  15. Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

    Science.gov (United States)

    Guo, Bingyuan; Sheng, Yingying; Zhou, Ke; Liu, Quansheng; Liu, Lei; Wu, Hai-Chen

    2018-03-26

    A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

    Science.gov (United States)

    Liu, Ting; Song, Menghuan; Xia, Yun; Zeng, Xiaomao

    2017-01-01

    In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. © 2017 S. Karger AG, Basel.

  17. Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil

    Energy Technology Data Exchange (ETDEWEB)

    Song, Mengke [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Jiang, Longfei [College of Life Sciences, Nanjing Agricultural University, Nanjing 210095 (China); Zhang, Dayi [Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ (United Kingdom); Luo, Chunling, E-mail: clluo@gig.ac.cn [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Wang, Yan [Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024 (China); Yu, Zhiqiang [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Yin, Hua [College of Environment and Energy, South China University of Technology, Guangzhou 510006 (China); Zhang, Gan [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China)

    2016-05-05

    Highlights: • Investigate PAHs degraders in forest carbon-rich soils via DNA-SIP. • Rhodanobacter is identified to metabolite anthracene for the first time. • The first fluoranthene degrader belongs to Acidobacteria. • Different functions of PAHs degraders in forest soils from contaminated soils. - Abstract: Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually {sup 13}C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

  18. Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

    Science.gov (United States)

    Woda, Marcia; Mathew, Anuja

    2015-01-01

    Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Elkin, Christopher; Kapur, Hitesh; Smith, Troy; Humphries, David; Pollard, Martin; Hammon, Nancy; Hawkins, Trevor

    2001-09-15

    We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620 phred20 bases as part of Joint Genome Institutes Production Process.

  20. An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

    Science.gov (United States)

    Chen, Sherry Xi; Seelig, Georg

    2016-04-20

    Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

  1. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Science.gov (United States)

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Immunophenotype of leukemic blasts with small peroxidase-positive granules detected by electron microscopy.

    Science.gov (United States)

    Vainchenker, W; Villeval, J L; Tabilio, A; Matamis, H; Karianakis, G; Guichard, J; Henri, A; Vernant, J P; Rochant, H; Breton-Gorius, J

    1988-05-01

    Forty-three cases of undifferentiated leukemias by light microscopy examination were diagnosed as acute myeloblastic leukemias by ultrastructural revelation of peroxidase and were subsequently studied by immunological markers. In 41 of these cases, blasts were labeled by at least one of the antimyeloid MoAbs (My 7, My 9, and 80H5). An antimyeloperoxidase polyclonal antibody was used in 23 cases and was clearly positive in 11 of them, while cytochemistry by light microscopy was negative. These myeloblasts were frequently mixed with a minority of blasts from other lineages especially promegakaryoblasts. It is noteworthy that in 6 cases myeloid and lymphoid markers (E rosette receptor, common acute lymphoblastic leukemia antigen (cALLA), CD 9, CD 19 antigens (anti-B4 MoAb] were detected on a fraction of blast cells, suggesting a bilineage leukemia. However, in double labeling experiments, blasts with myeloperoxidase coexpressed lymphoid and myeloid markers including cALLA and CD 19 antigen. In one case, blasts had a typical non-B, non-T acute lymphoblastic leukemia phenotype (HLA-DR, CD 9, CD 19, cALLA positive) without staining by any of the antimyeloid MoAbs. However, 70% of the blasts were labeled by the antimyeloperoxidase antibody and expressed peroxidase-positive granules at ultrastructural level. In conclusion, most of the AML undiagnosed by optical cytochemistry are identified by antimyeloid antibodies. Some of these cases are also stained by some antilymphoid MoAbs. Use of antibodies against myeloperoxidase may improve the diagnosis of difficult cases of acute myeloblastic leukemia.

  3. Influence of Divalent Counterions on the Dynamics in DNA as Probed by Using a Minor-Groove Binder.

    Science.gov (United States)

    Paul, Sneha; Ahmed, Tasnim; Samanta, Anunay

    2017-08-05

    DNA dynamics, to which water, counterions, and DNA motions contribute, is a topic of considerable interest because it is closely related to the efficiency of biological functions performed by it. Simulation studies and experiments suggest that the counterion dynamics in DNA probed by a minor-groove binder are similar for various monovalent counterions. To date, the influence on DNA dynamics of higher-valence counterions, which are also present around DNA and are known to bind more strongly to it than monovalent ions, has not been studied. Herein we investigated DNA dynamics in the presence of Mg 2+ and Ca 2+ , chosen for their relative abundance in cells, by using minor-groove binder 4',6-diamidino-2-phenylindole (DAPI) as a fluorescence probe. The dynamics, as measured from the time-resolved fluorescence Stokes shifts of DAPI bound to calf thymus DNA on a subpicosecond-to-nanosecond timescale, were found to be very similar in the presence of both the divalent ions and Na + ions. The observation is explained by considering the screening of the electric field of the divalent ion by its hydration shell, preferential binding of the ions to the phosphate groups, and displacement of ions from the minor groove by DAPI due to the stronger binding interaction of the latter. Furthermore, the similarity of our results in the presence of Na + to those reported for smaller oligonucleotides suggests that the chain length of DNA does not influence the DNA dynamics. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Armelle Cabin-Flaman

    2016-06-01

    Full Text Available Dynamic secondary ion mass spectrometry (D-SIMS imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C14N- recombinant ion and the use of the 13C:12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS.

  5. Neocarzinostatin as a probe for DNA protection activity--molecular interaction with caffeine.

    Science.gov (United States)

    Chin, Der-Hang; Li, Huang-Hsien; Kuo, Hsiu-Maan; Chao, Pei-Dawn Lee; Liu, Chia-Wen

    2012-04-01

    Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role. Copyright © 2011 Wiley Periodicals, Inc.

  6. A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

    International Nuclear Information System (INIS)

    Gendelman, H.E.; Moench, T.R.; Narayan, O.; Griffin, D.E.; Clements, J.E.

    1985-01-01

    This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). This technique provides a new approach to the study of viral pathogenesis by: (1) identifying the types of cells which are infected in the host and (2) identifying points of blockade in the virus life cycle during persistent infections. (Auth.)

  7. Probing the Structure of DNA Aptamers with a Classic Heterocycle.

    Directory of Open Access Journals (Sweden)

    G. Reid Bishop

    2004-02-01

    Full Text Available DNA aptamers are synthetic, single-stranded DNA oligonucleotides selectedby SELEX methods for their binding with specific ligands. Here we present ethidiumbinding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARGthat bind L-argininamide (L-Arm. The ligand bound form of each aptamer's structurehas been reported and each are found to be composed primarily of two domainsconsisting of a stem helical region and a loop domain that forms a binding pocket for thecognate ligand. Previous thermodynamic experiments demonstrated that the DNAaptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Herewe extend those linkage binding studies by examining the binding of the heterocyclicintercalator ethidium to each of the three aptamers by fluorescence and absorptionspectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with∆Go's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for eachaptamer and is quantitatively diminished in the presence of L-Arm as is the overallfluorescence intensity of ethidium. Together, these results demonstrate that a portion ofthe bound ethidium is excluded from the aptamer in the presence of a saturating amountof L-Arm. These results demonstrate the utility of ethidium and related compounds forthe probing of non-conventional DNA structures and reveal an interesting fundamentalthermodynamic linkage in DNA aptamers. Results are discussed in the context of thethermodynamic stability and structure of each of the aptamers examined.

  8. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

    Directory of Open Access Journals (Sweden)

    Nidhi Mathur

    2008-01-01

    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  9. Biconically Tapered Fiber Optic Probes for Rapid Label-Free Immunoassays

    Directory of Open Access Journals (Sweden)

    John Miller

    2015-04-01

    Full Text Available We report use of U-shaped biconically tapered optical fibers (BTOF as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G-antigen (rabbit anti-mouse IgG reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

  10. Synthesis of novel fluorescent probe Tb(III)-7-carboxymethoxy-4-methylcoumarin complex for sensing of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hussein, Belal H.M., E-mail: belalhussein102@yahoo.com [Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia (Egypt); Azab, Hassan A. [Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia (Egypt); Fathalla, Walid [Department of Mathematical and Physical Sciences, Faculty of Engineering, Port-Said University, Port-Said (Egypt); Ali, Sherin A.M. [Department of Mathematical and Physical Sciences, Faculty of Engineering, Suez Canal University, Ismailia (Egypt)

    2013-02-15

    New fluorescent probe Tb(III) (7-carboxymethoxy-4-methylcoumarin)2(SCN) (C2H5OH)(H2O) was synthesized and characterized by spectroscopy and thermal analysis. The absorption and fluorescence spectra of 7-carboxymethoxy-4-methylcoumarin (CMMC) and Tb(III)-CMMC complex have been measured in different solvents. The interactions of Tb(III)-CMMC complex with calf thymus nucleic acid (CT-DNA) have been investigated using steady state fluorescence measurements. The changes in the fluorescence intensity have been used for the quantitative determination of DNA with LOD of 3.45 ng in methanol-water (9:1, v/v). The association constants of DNA with Tb(III)-CMMC complex was found to be 2.62 Multiplication-Sign 1010 M{sup -1}. - Highlights: Black-Right-Pointing-Pointer New fluorescent probe Terbium (III)-7-carboxy methoxy-4-methylcoumarin complex has been synthesized and characterized. Black-Right-Pointing-Pointer FTIR spectrum of Tb(III)-complex shows a characteristic band for thiocyanate group. Black-Right-Pointing-Pointer DNA interaction with Terbium (III)-7-carboxy methoxy-4-methylcoumarin has been studied by fluorescence techniques. Black-Right-Pointing-Pointer The change in the fluorescence intensity has been used for the quantitative determination of DNA. Black-Right-Pointing-Pointer The result was better than most of the well-known methods including the ethidium bromide method.

  11. One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2016-10-26

    Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were

  12. Magnetoresistive sensors for measurements of DNA hybridization kinetics - effect of TINA modifications

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt

    2017-01-01

    We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current....... A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic...

  13. Regional localization of DNA probes on the short arm of chromosome 11 using aniridia-Wilms' tumor-associated deletions

    NARCIS (Netherlands)

    Mannens, M.; Slater, R. M.; Heyting, C.; Geurts van Kessel, A.; Goedde-Salz, E.; Frants, R. R.; van Ommen, G. J.; Pearson, P. L.

    1987-01-01

    We are interested in the precise localization of various DNA probes on the short arm of chromosome 11 for our research on the aniridia-Wilms' tumor association (AWTA), assigned to region 11p13 (Knudson and Strong 1972; Riccardi et al. 1978). For this purpose we have screened lymphocyte DNA and

  14. A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus.

    Science.gov (United States)

    Decaro, Nicola; Elia, Gabriella; Desario, Costantina; Roperto, Sante; Martella, Vito; Campolo, Marco; Lorusso, Alessio; Cavalli, Alessandra; Buonavoglia, Canio

    2006-09-01

    A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.

  15. Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA

    Directory of Open Access Journals (Sweden)

    Chan Alan

    2006-06-01

    Full Text Available Abstract Background Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. Results In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A. Conclusion Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

  16. Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA.

    Science.gov (United States)

    Gibbons, Brian; Datta, Parikkhit; Wu, Ying; Chan, Alan; Al Armour, John

    2006-06-30

    Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH) we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A). Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

  17. DNA nanosensor based on biocompatible graphene quantum dots and carbon nanotubes.

    Science.gov (United States)

    Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Ma, Juan Juan; Chen, Jian Rong; Feng, Hui

    2014-10-15

    An ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) between biocompatible graphene quantum dots and carbon nanotubes for DNA detection was reported. We take advantage of good biocompatibility and strong fluorescence of graphene quantum dots, base pairing specificity of DNA and unique fluorescence resonance energy transfer between graphene quantum dots and carbon nanotubes to achieve the analysis of low concentrations of DNA. Graphene quantum dots with high quantum yield up to 0.20 were prepared and served as the fluorophore of DNA probe. FRET process between graphene quantum dots-labeled probe and oxidized carbon nanotubes is easily achieved due to their efficient self-assembly through specific π-π interaction. This nanosensor can distinguish complementary and mismatched nucleic acid sequences with high sensitivity and good reproducibility. The detection method based on this nanosensor possesses a broad linear span of up to 133.0 nM and ultralow detection limit of 0.4 nM. The constructed nanosensor is expected to be highly biocompatible because of all its components with excellent biocompatibility. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c

    Science.gov (United States)

    Muenzner, Julia; Toffey, Jason R.; Hong, Yuning; Pletneva, Ekaterina V.

    2014-01-01

    Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes. PMID:23713573

  19. Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints.

    Science.gov (United States)

    Heuer, H; Hartung, K; Wieland, G; Kramer, I; Smalla, K

    1999-03-01

    Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5' exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

  20. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    Science.gov (United States)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  1. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  2. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Departamento de Parasitologia. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-07-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with {sup 32}P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  3. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    International Nuclear Information System (INIS)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R.

    2011-01-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with 32 P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  4. Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection.

    Directory of Open Access Journals (Sweden)

    Yingwei Zhang

    Full Text Available In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs assembled from Cu(II and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1 RCPN binds dye-labeled single-stranded DNA (ssDNA probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2 Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.

  5. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  6. Multilocus DNA fingerprinting in paternity analysis: a Chilean experience

    Directory of Open Access Journals (Sweden)

    Cifuentes O. Lucía

    2000-01-01

    Full Text Available DNA polymorphism is very useful in paternity analysis. The present paper describes paternity studies done using DNA profiles obtained with the (CAC5 probe. All of the subjects studied were involved in nonjudicial cases of paternity. Genomic DNA digested with HaeIII was run on agarose gels and hybridized in the gel with the (CAC5 probe labeled with 32P. The mean number of bands larger than the 4.3 kb per individual was 16.1. The mean proportion of bands shared among unrelated individuals was 0.08 and the mean number of test bands was 7.1. This corresponded to an exclusion probability greater than 0.999999. Paternity was excluded in 34.5% of the cases. The mutation frequency estimated from non-excluded cases was 0.01143 bands per child. In these cases, the paternity was confirmed by a locus-specific analysis of eight independent PCR-based loci. The paternity index was computed in all non-excluded cases. It can be concluded that this method is a powerful and inexpensive alternative to solve paternity doubts.

  7. Low-cost label-free electrical detection of artificial DNA nanostructures using solution-processed oxide thin-film transistors.

    Science.gov (United States)

    Kim, Si Joon; Jung, Joohye; Lee, Keun Woo; Yoon, Doo Hyun; Jung, Tae Soo; Dugasani, Sreekantha Reddy; Park, Sung Ha; Kim, Hyun Jae

    2013-11-13

    A high-sensitivity, label-free method for detecting deoxyribonucleic acid (DNA) using solution-processed oxide thin-film transistors (TFTs) was developed. Double-crossover (DX) DNA nanostructures with different concentrations of divalent Cu ion (Cu(2+)) were immobilized on an In-Ga-Zn-O (IGZO) back-channel surface, which changed the electrical performance of the IGZO TFTs. The detection mechanism of the IGZO TFT-based DNA biosensor is attributed to electron trapping and electrostatic interactions caused by negatively charged phosphate groups on the DNA backbone. Furthermore, Cu(2+) in DX DNA nanostructures generates a current path when a gate bias is applied. The direct effect on the electrical response implies that solution-processed IGZO TFTs could be used to realize low-cost and high-sensitivity DNA biosensors.

  8. Rapid and inexpensive method for isolating plasmid DNA

    International Nuclear Information System (INIS)

    Aljanabi, S. M.; Al-Awadi, S. J.; Al-Kazaz, A. A.; Baghdad Univ.

    1997-01-01

    A small-scale and economical method for isolating plasmid DNA from bacteria is described. The method provides DNA of suitable quality for most DNA manipulation techniques. This DNA can be used for restriction endonuclease digestion, southern blot hybridization, nick translation and end labeling of DNA probes, Polymerase Chain Reaction (PCR) -based techniques, transformation, DNA cycle-sequencing, and Chain-termination method for DNA sequencing. The entire procedure is adapted to 1.5 ml microfuge tubes and takes approximately 30 mins. The DNA isolated by this method has the same purity produced by CTAB and cesium chloride precipitation and purification procedures respectively. The two previous methods require many hours to obtain the final product and require the use of very expensive equipment as ultracentrifuge. This method is well suited for the isolation of plasmid DNA from a large number of bacterial samples and in a very short time and low cost in laboratories where chemicals, expensive equipment and finance are limited factors in conducting molecular research. (authors). 11refs. 11refs

  9. Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptide-major histocompatibility complex I multimers

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

    2016-01-01

    sample using >1000 different peptide-MHC multimers labeled with individual DNA barcodes.After isolation of MHC multimer binding T cells their recognition are revealed by amplification andsequencing of the MHC multimer-associated DNA barcodes. The relative frequency of the sequencedDNA barcodes...... originating from a given peptide-MHC motif relates to the size of the antigenresponsiveT cell population. We have demonstrated the use of large panels of >1000 DNA barcodedMHC multimers for detection of rareT cell populations of virus and cancer-restricted origin in various tissues and compared...

  10. Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila

    International Nuclear Information System (INIS)

    Tschunko, A.H.; Loechel, R.H.; McLaren, N.C.; Allen, S.L.

    1987-01-01

    DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. Inverted repeated sequences were present in one clone. This clone, pTtFBl, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. DNA was labeled with [ 33 P]-labeled dATP. The authors found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat are totally eliminated during macronuclear development-and contain open reading frames. The significance of retained inverted repeats to the process of elimination is discussed

  11. DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

    Science.gov (United States)

    Plazinski, Jacek; Zheng, Qi; Taylor, Rona; Croft, Lynn; Rolfe, Barry G.; Gunning, Brian E. S.

    1990-01-01

    Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp. Images PMID:16348182

  12. High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

    Science.gov (United States)

    Andeer, Peter; Strand, Stuart E; Stahl, David A

    2012-01-01

    Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

  13. Organic liquids-responsive β-cyclodextrin-functionalized graphene-based fluorescence probe: label-free selective detection of tetrahydrofuran.

    Science.gov (United States)

    Hu, Huawen; Xin, John H; Hu, Hong; Wang, Xiaowen; Lu, Xinkun

    2014-06-06

    In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD) was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB) into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  14. Organic Liquids-Responsive β-Cyclodextrin-Functionalized Graphene-Based Fluorescence Probe: Label-Free Selective Detection of Tetrahydrofuran

    Directory of Open Access Journals (Sweden)

    Huawen Hu

    2014-06-01

    Full Text Available In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  15. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism.

    Science.gov (United States)

    Weis, V M; Small, A L; McFall-Ngai, M J

    1996-11-26

    Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking. In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri. Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils. MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ. We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity. An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis. These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.

  16. A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

    Science.gov (United States)

    Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

    2018-04-23

    Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Directory of Open Access Journals (Sweden)

    P. Schendzielorz

    2016-01-01

    Full Text Available Adipose-derived stem cells (ASCs have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342 represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL, with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

  18. Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

    International Nuclear Information System (INIS)

    Xiang Xia; Luo Ming; Shi Liyang; Ji Xinghu; He Zhike

    2012-01-01

    Graphical abstract: With a microvalve manipulate technique combined with droplet platform, a microscale fluorescence-based colorimetric sensor for multiplexed DNA analysis is developed via a graphene nanoprobe. Highlights: ► A quantitative detection for multiplexed DNA is first realized on droplet platform. ► The DNA detection is relied on a simple fluorescence-based colorimetric method. ► GO is served as a quencher for two different DNA fluorescent probes. ► This present work provides a rapid, sensitive, visual and convenient detection tool for droplet biosensor. - Abstract: The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10 −8 M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.

  19. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  20. A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

    Science.gov (United States)

    Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

    2017-01-01

    A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

  1. New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

    Science.gov (United States)

    Gbaj, A; Bichenkova, Ev; Walsh, L; Savage, He; Sardarian, Ar; Etchells, Ll; Gulati, A; Hawisa, S; Douglas, Kt

    2009-12-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

  2. Poly(o-phenylenediamine) colloid-quenched fluorescent oligonucleotide as a probe for fluorescence-enhanced nucleic acid detection.

    Science.gov (United States)

    Tian, Jingqi; Li, Hailong; Luo, Yonglan; Wang, Lei; Zhang, Yingwei; Sun, Xuping

    2011-02-01

    In this Letter, we demonstrate that chemical oxidation polymerization of o-phenylenediamine (OPD) by potassium bichromate at room temperature results in the formation of submicrometer-scale poly(o-phenylenediamine) (POPD) colloids. Such colloids can absorb and quench dye-labeled single-stranded DNA (ssDNA) very effectively. In the presence of a target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. With the use of an oligonucleotide (OND) sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch.

  3. Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

    Science.gov (United States)

    Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2013-01-01

    A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

  4. Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

    Science.gov (United States)

    Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

    2011-06-01

    This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

  5. Molecular verification on male sterile mutant after injected exogenous λDNA into wheat

    International Nuclear Information System (INIS)

    Yang Jingcheng; Yu Yuanjie; Liu Fengzhen; Qi Yanfang; Shen Fafu

    2000-01-01

    A cytoplasmic male sterile mutant and then a stable CMS line named D-type sterile line were obtained after injected exogenous λDNA into wheat line 814527, and line 814527 could be its maintainer line. By using λDNA labelled with 32 P as probe, unlabelled λDNA as positive check, dot blotting of nuclear DNA and chloroplast DNA of receptor 814527, D-type sterile line and its hybrid F 1 with Lumai 14 were carried out. Positive dots appeared in nuclear DNA and chloroplast DNA of D-type sterile line and its hybrid F 1 , but did not appear in the receptor. It showed that fragments of exogenous λDNA existed in nuclear genome and chloroplast genome of D-type sterile line, and could be inherited stably. All these results, on a molecular level, proved the reliability of exogenous DNA injection

  6. Silencing of glutathione peroxidase 3 through DNA hypermethylation is associated with lymph node metastasis in gastric carcinomas.

    Directory of Open Access Journals (Sweden)

    Dun-Fa Peng

    Full Text Available Gastric cancer remains the second leading cause of cancer-related death in the world. H. pylori infection, a major risk factor for gastric cancer, generates high levels of reactive oxygen species (ROS. Glutathione peroxidase 3 (GPX3, a plasma GPX member and a major scavenger of ROS, catalyzes the reduction of hydrogen peroxide and lipid peroxides by reduced glutathione. To study the expression and gene regulation of GPX3, we examined GPX3 gene expression in 9 gastric cancer cell lines, 108 primary gastric cancer samples and 45 normal gastric mucosa adjacent to cancers using quantitative real-time RT-PCR. Downregulation or silencing of GPX3 was detected in 8 of 9 cancer cell lines, 83% (90/108 gastric cancers samples, as compared to non-tumor adjacent normal gastric samples (P<0.0001. Examination of GPX3 promoter demonstrated DNA hypermethylation (≥ 10% methylation level determined by Bisulfite Pyrosequencing in 6 of 9 cancer cell lines and 60% of gastric cancer samples (P = 0.007. We also detected a significant loss of DNA copy number of GPX3 in gastric cancers (P<0.001. Treatment of SNU1 and MKN28 cells with 5-Aza-2' Deoxycytidine restored the GPX3 gene expression with a significant demethylation of GPX3 promoter. The downregulation of GPX3 expression and GPX3 promoter hypermethylation were significantly associated with gastric cancer lymph node metastasis (P = 0.018 and P = 0.029, respectively. We also observed downregulation, DNA copy number losses, and promoter hypermethylation of GPX3 in approximately one-third of tumor-adjacent normal gastric tissue samples, suggesting the presence of a field defect in areas near tumor samples. Reconstitution of GPX3 in AGS cells reduced the capacity of cell migration, as measured by scratch wound healing assay. Taken together, the dysfunction of GPX3 in gastric cancer is mediated by genetic and epigenetic alterations, suggesting impairment of mechanisms that regulate ROS and its possible involvement in

  7. Genetic effect of A-bomb radiation- Analysis of minisatellite regions detected by DNA fingerprint probe

    International Nuclear Information System (INIS)

    Kodaira, Mieko

    1999-01-01

    In author's laboratory, screening of mutation in germ cells of A-bomb survivors is under investigation with use of 8 single-locus minisatellite probes and no increase in mutation rate has been detected hitherto. This paper reported results of screening on the minisatellite region, which consisting of short repeated base sequence, using a DNA fingerprint probe for 33.15 core sequence. Subjects were 50 A-bomb survivor families exposed to mean dose of 1.9 Sv (exposed group) or 0 Gy (control), having 64 or 60 children, respectively. DNA was extracted from their B cells established by EB virus and subjected to agarose-gel electrophoresis followed by southern blotting with some improvements for fingerprinting. On the fingerprints, numbers of the band detected in regions of >3.5 kb were 1080 in children of the exposed group (16.9/child) and 1024 (17.1) in the control group, indicating no detectable effect of exposure on the germ cell mutation rate in the region.(K.H.)

  8. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    Science.gov (United States)

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Peroxidase activity as a marker for estrogenicity

    International Nuclear Information System (INIS)

    Levy, J.; Liel, Y.; Glick, S.M.

    1981-01-01

    We examined the possibility that peroxidase activity might be a marker for estrogen activity in established estrogen-dependent tissues: dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumours and human breast cancer. In DMBA-induced tumours undergoing regression after ovariectomy or tamoxifen treatment, tumour size decreased by 50%, estradiol receptors (ER) and progesterone receptors (PgR) decreased by 25 and 20%, respectively, but peroxidase activity paradoxically increased six- to sevenfold. In DMBA tumours stimulated by estradiol treatment or by the cessation of tamoxifen administration in intact rats, tumour size increased threefold. ER and PgR increased two- and threefold, respectively, while peroxidase activity decreased 50%. These data indicate an inverse relation between tumour growth, ER and PgR on the one hand, and peroxidase activity on the other. In the human breast cancers there was a singificant negative relation between the presence of ER and peroxidase activity. By using a calibrated Sephadex G-100 column it was shown that uterine peroxidase differs in molecular weight from the peroxidase of rat mammary tumours and that of human breast cancer. (author)

  10. Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

    Science.gov (United States)

    Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

    2008-04-01

    The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

  11. Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

    Science.gov (United States)

    Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

    2015-02-01

    In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

  12. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

    Science.gov (United States)

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

    2015-01-01

    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  13. New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

    Science.gov (United States)

    Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

    2012-01-01

    Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

  14. DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

    Science.gov (United States)

    Sun, Feifei; You, Ying; Liu, Jie; Song, Quanwei; Shen, Xiaotong; Na, Na; Ouyang, Jin

    2017-05-23

    A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Oxidation of NAD dimers by horseradish peroxidase.

    OpenAIRE

    Avigliano, L; Carelli, V; Casini, A; Finazzi-Agrò, A; Liberatore, F

    1985-01-01

    Horseradish peroxidase catalyses the oxidation of NAD dimers, (NAD)2, to NAD+ in accordance with a reaction that is pH-dependent and requires 1 mol of O2 per 2 mol of (NAD)2. Horseradish peroxidase also catalyses the peroxidation of (NAD)2 to NAD+. In contrast, bacterial NADH peroxidase does not catalyse the peroxidation or the oxidation of (NAD)2. A free-radical mechanism is proposed for both horseradish-peroxidase-catalysed oxidation and peroxidation of (NAD)2.

  16. Fluorescent Oligonucleotides Containing a 2-Ethynylfluorene-or 2-Ethynylfluorenone-labeled 2'-Deoxyguanosine Unit: Fluorescence Changes upon Duplex Formation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min Ji; Hwang, Gil Tae [Kyungpook National University, Daegu (Korea, Republic of)

    2016-08-15

    Two new DNA probes bearing a fluorescent deoxyguanosine unit labeled with 2-ethynylfluorene (G{sup FL} )or 2-ethynylfluorenone (G{sup FO}) were synthesized and examined for their efficiency as quencher-free linear beacon probes. Oligodeoxynucleotides (ODNs) containing a G{sup FL} or G{sup FO} unit exhibit low thermal selectivity and few distinctive fluorescence changes upon duplex formation due to the syn conformation about the glycosidic bond. An exciplex emission was observed when the G{sup FL} unit of ODNs bearing adenine flanking bases was positioned opposite to the adenine nucleobases.

  17. Uses of Dendrimers for DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Majoral

    2006-08-01

    Full Text Available Biosensors such as DNA microarrays and microchips are gaining an increasingimportance in medicinal, forensic, and environmental analyses. Such devices are based onthe detection of supramolecular interactions called hybridizations that occur betweencomplementary oligonucleotides, one linked to a solid surface (the probe, and the other oneto be analyzed (the target. This paper focuses on the improvements that hyperbranched andperfectly defined nanomolecules called dendrimers can provide to this methodology. Twomain uses of dendrimers for such purpose have been described up to now; either thedendrimer is used as linker between the solid surface and the probe oligonucleotide, or thedendrimer is used as a multilabeled entity linked to the target oligonucleotide. In the firstcase the dendrimer generally induces a higher loading of probes and an easier hybridization,due to moving away the solid phase. In the second case the high number of localized labels(generally fluorescent induces an increased sensitivity, allowing the detection of smallquantities of biological entities.

  18. N-(2-chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with 32P-end-labeled DNA

    International Nuclear Information System (INIS)

    Church, K.M.; Wurdeman, R.L.; Zhang, Yi; Chen, Faxian; Gold, B.

    1990-01-01

    The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in 32 P-end-labeled restriction fragments with methidiumpropyl-EDTA·Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using 32 P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. Linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion

  19. Automated DNA electrophoresis, hybridization and detection

    International Nuclear Information System (INIS)

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-01-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; 32 P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing

  20. A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

    Science.gov (United States)

    Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

    2012-01-01

    We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

  1. Norrie disease: linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe.

    Science.gov (United States)

    Ngo, J T; Bateman, J B; Cortessis, V; Sparkes, R S; Mohandas, T; Inana, G; Spence, M A

    1989-05-01

    Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.

  2. Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS.

    Science.gov (United States)

    Li, Xiaoting; Chen, Beibei; He, Man; Xiao, Guangyang; Hu, Bin

    2018-01-01

    In this work, we developed an immunoassay based on tyramide signal amplification (TSA) and gold nanoparticles (Au NPs) labeling for highly sensitive detection of alpha fetoprotein (AFP) by inductively coupled plasma mass spectrometry (ICP-MS). AFP was captured by anti-AFP1 coating on the 96-well plate and labeled by anti-AFP2-horseradish peroxidase (HRP), in which the HRP can catalyze the deposition of biotinylated tyramine on the nearby protein. Then the streptavidin (SA)-Au NPs was labeled on the deposited biotinylated tyramine as the intensive signal probe for ICP-MS measurement. Under the optimal experimental conditions, the limit of detection of the developed method for AFP was 1.85pg/mL and the linear range was 0.005-2ng/mL. The relative standard deviation for seven replicate detections of 0.01ng/mL AFP was 5.2%. The proposed method was successfully applied to the detection of AFP in human serum with good recoveries. This strategy is highly sensitive and easy to operate, and can be extended to the sensitive detection of other biomolecules in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs

    Directory of Open Access Journals (Sweden)

    Eva Vargas

    2017-11-01

    Full Text Available This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs. The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21 with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs, and labeling of the resulting heteroduplexes with a specific DNA–RNA antibody and the bacterial protein A (ProtA conjugated with an horseradish peroxidase (HRP homopolymer (Poly-HRP40 as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs using the H2O2/hydroquinone (HQ system. The magnitude of the cathodic signal obtained at −0.20 V (vs. the Ag pseudo-reference electrode demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD of 10 attomoles (in a 25 μL sample without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared. This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA–RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNAt extracted from human mammary epithelial normal (MCF-10A and cancer (MCF-7 cells and tumor tissues.

  4. Development and Characterization of Complex DNA Fingerprinting Probes for the Infectious Yeast Candida dubliniensis

    Science.gov (United States)

    Joly, Sophie; Pujol, Claude; Rysz, Michal; Vargas, Kaaren; Soll, David R.

    1999-01-01

    Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast Candida dubliniensis, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of Candida albicans. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the C. dubliniensis genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly C. dubliniensis-specific probe of the three, generated the most complex pattern, was distributed throughout all C. dubliniensis chromosomes, and separated a worldwide collection of 57 C. dubliniensis isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that C. dubliniensis represents a bona fide species. PMID:10074523

  5. "Chitin-specific" peroxidases in plants.

    Science.gov (United States)

    Maksimov, I V; Cherepanova, E A; Khairullin, R M

    2003-01-01

    The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega(Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.

  6. Simple synthesis of carbon-11-labeled chromen-4-one derivatives as new potential PET agents for imaging of DNA-dependent protein kinase (DNA-PK) in cancer

    International Nuclear Information System (INIS)

    Gao, Mingzhang; Wang, Min; Miller, Kathy D.; Zheng, Qi-Huang

    2012-01-01

    Carbon-11-labeled chromen-4-one derivatives were synthesized as new potential PET agents for imaging of DNA repair enzyme DNA-dependent protein kinase (DNA-PK) in cancer. The target tracers, X-[ 11 C]methoxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; [ 11 C]4a–d), were prepared from their corresponding precursors, X-hydroxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; 5a–d), with [ 11 C]CH 3 OTf through O-[ 11 C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a C-18 Sep-Pak Plus cartridge. The radiochemical yields decay corrected to end of bombardment (EOB), from [ 11 C]CO 2 , were 40–60%. The specific activity at end of synthesis (EOS) was 185–370 GBq/μmol. - Highlights: ► New chromen-4-one derivatives were synthesized. ► New carbon-11-labeled chromen-4-one derivatives were synthesized. ► Simple solid-phase extraction (SPE) method was employed in radiosynthesis.

  7. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-01

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little...

  8. The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

    Directory of Open Access Journals (Sweden)

    Abrenica Maria V

    2010-09-01

    Full Text Available Abstract Background Mycobacterium tuberculosis topoisomerase I (MtTOP1 and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex. Results We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate. Conclusions The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

  9. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Datta, K.; Dizdaroglu, M.; Jaruga, P.; Neumann, R.D.; Winters, T.A.

    2003-01-01

    Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125 I in a plasmid bound by a 125 I-labeled triplex forming oligonucleotide ( 125 I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125 I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125 Itarget base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the absence

  10. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-01

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  11. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    Directory of Open Access Journals (Sweden)

    Gbaj A

    2009-01-01

    Full Text Available The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5’-bispyrene and 3’-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5’-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

  12. Label-free detection of DNA hybridization using transistors based on CVD grown graphene.

    Science.gov (United States)

    Chen, Tzu-Yin; Loan, Phan Thi Kim; Hsu, Chang-Lung; Lee, Yi-Hsien; Tse-Wei Wang, Jacob; Wei, Kung-Hwa; Lin, Cheng-Te; Li, Lain-Jong

    2013-03-15

    The high transconductance and low noise of graphene-based field-effect transistors based on large-area monolayer graphene produced by chemical vapor deposition are used for label-free electrical detection of DNA hybridization. The gate materials, buffer concentration and surface condition of graphene have been optimized to achieve the DNA detection sensitivity as low as 1 pM (10(-12) M), which is more sensitive than the existing report based on few-layer graphene. The graphene films obtained using conventional PMMA-assisted transfer technique exhibits PMMA residues, which degrade the sensing performance of graphene. We have demonstrated that the sensing performance of the graphene samples prepared by gold-transfer is largely enhanced (by 125%). Copyright © 2012 Elsevier B.V. All rights reserved.

  13. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

    Science.gov (United States)

    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

    Science.gov (United States)

    Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

    2012-01-01

    The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

  15. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  16. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

    2015-01-01

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...

  17. Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

    DEFF Research Database (Denmark)

    Henriksen, A; Mirza, O; Indiani, C

    2001-01-01

    Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous...... glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact...... with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could...

  18. Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

    International Nuclear Information System (INIS)

    Cekan, Pavol; Sigurdsson, Snorri Th.

    2012-01-01

    Highlights: ► Bulges and loops were studied by both EPR and fluorescence spectroscopies using the probe Ç/Ç f . ► One-base bulge was in a temperature-dependent equilibrium between looped-out and stacked states. ► Bases in two- and three-base bulges were stacked at all temperatures, resulting in DNA bending. ► Bases were stacked in symmetrical two- to five-base internal loops, according to EPR data. ► Unexpectedly high fluorescence for the smaller loops indicated local structural perturbations. -- Abstract: The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe Ç, which becomes fluorescent (Ç f ) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of Ç f . EPR spectra of Ç-labeled three-, four- and five-base symmetrical interior DNA bulges at 20 °C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37 °C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2 °C. Fluorescence of the smallest loops, where a single T·T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops from regular B-DNA. These results show the value of combining EPR and fluorescence spectroscopy to study non-helical regions of nucleic acids.

  19. Fluorographic determination of /sup 3/H-labelled hepatitis B virus DNA and other viral DNAs

    Energy Technology Data Exchange (ETDEWEB)

    Jantschak, J; Meisel, H

    1986-01-01

    The radioactivity of aqueous samples of /sup 3/H-labelled DNA can easily be determined by a quantitative fluorographic assay. 25 samples were collected and fixed simultaneously onto a membrane filter. Their fluorographic film blackening is compared densitometrically or visually to that of standard samples. The lower limit of detection is 1.7 Bq (100 dpm). This procedure is very effective when dealing with high numbers of samples and presents an economically favorable alternative to scintillation counting. The efficiency of the method for the assay of different DNA polymerase activities (hepatitis B virus, bovine leukemia virus, bacteriophage T 4) could be demonstrated.

  20. Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

    Science.gov (United States)

    Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

    2012-04-25

    A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.