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Sample records for oligonucleotide probe hybridization

  1. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    Science.gov (United States)

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  2. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

    Science.gov (United States)

    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

  3. Fluoroscence in situ hybridization of chicken intestinal samples with bacterial rRNA targeted oligonucleotide probes

    DEFF Research Database (Denmark)

    Olsen, Katja Nyholm; Francesch, M.; Christensen, Henrik

    2006-01-01

    The objective was to develop a fast and accurate molecular method for the quantification of the intestinal flora in chickens by rRNA fluorescence in situ hybridization (FISH). Seven weeks old conventionally reared Lohmann hens were used to set up the method. To sample ileal intestinal content......, the distal part from Meckels diverticulum to the ileo-caecal junction was removed. Fixation was performed in ethanol and phosphate buffered saline. After washing by centrifugation, the sample was resuspended in pre-heated hybridization buffer with oligonucleotide probe labelled with Cy3 (10ng/µl). The cells...... were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB...

  4. 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.

  5. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

    1988-11-01

    Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with /sup 32/P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

  6. Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

    OpenAIRE

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-01-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a C...

  7. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

    Science.gov (United States)

    Hakala, H; Heinonen, P; Iitiä, A; Lönnberg, H

    1997-01-01

    Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

  8. Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    DEFF Research Database (Denmark)

    Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

    1999-01-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas, Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid...... fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria-and methanogens were compared to specific methanogenic activities...

  9. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  10. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  11. Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization.

    Science.gov (United States)

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-08-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

  12. Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    DEFF Research Database (Denmark)

    Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

    1999-01-01

    -beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature of-dissociation and specificity studies, To demonstrate the usefulness of the probes for the detection and quantification of saturated......Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria-and methanogens were compared to specific methanogenic activities...

  13. Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

    Science.gov (United States)

    Oerther, D B; de los Reyes, F L; de los Reyes, M F; Raskin, L

    2001-10-01

    Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

  14. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  15. Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Sakai, Kenji; Mori, Masatsugu; Fujii, Akira; Iwami, Yuko; Chukeatirote, Ekachai; Shirai, Yoshihito

    2004-01-01

    Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.

  16. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    Directory of Open Access Journals (Sweden)

    Jennifer G Mulle

    Full Text Available DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs, and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  17. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    Science.gov (United States)

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  18. Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

    followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, a viral RNA sequence can be detected in less than 2 h without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity of ... opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  19. An oligonucleotide hybridization approach to DNA sequencing.

    Science.gov (United States)

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D

    1989-10-09

    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  20. Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota.

    Science.gov (United States)

    Momose, Y; Park, S H; Miyamoto, Y; Itoh, K

    2011-07-01

    To develop species-specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. The specificity of oligonucleotide probes was evaluated by fluorescence whole-cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides-Parabacteroides microbiota of conventional mice and specific pathogen-free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group-1, Group-2 and Group-3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides-Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides-Parabacteroides microbiota. The Group-3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group-1 and Group-2. The species-specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides-Parabacteroides community. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  1. One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection

    Directory of Open Access Journals (Sweden)

    Tinawi-Aljundi R

    2015-04-01

    Full Text Available Rima Tinawi-Aljundi,1 Lauren King,2 Shannon T Knuth,2 Michael Gildea,2 Carrie Ng,2 Josh Kahl,2 Jacqueline Dion,2 Chris Young,2 Edward W Schervish,1 J Rene Frontera,1 Jason Hafron,1 Kenneth M Kernen,1 Robert Di Loreto,1 Joan Aurich-Costa21Michigan Institute of Urology, St Claire Shores, MI, USA; 2Cellay, Inc., Cambridge, MA, USA Background: Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. Materials and methods: In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. Results: Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%–94.7% accuracy, 96.8% sensitivity (95% CI: 91.0%–99.3%, 79.2% specificity (95% CI: 65.9%–87.8%, 89.2% positive predictive value (PPV; 95% CI: 81.5%–94.5%, and 93.3% negative predictive value (NPV; 95% CI: 81.7%–97.3%. When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%–88.5%, 82.0% sensitivity (95% CI: 76.0%–87.1%, 88.4% specificity (95% CI: 83.2%–92.5%, 87.7% PPV (95% CI: 82.1%–92.0%, and 83.0% NPV (95% CI: 77.3%–87.8%. When looking at low- and high-grade tumors, the test showed 100

  2. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    Science.gov (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  3. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

    Science.gov (United States)

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V

    2009-10-01

    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  4. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    Science.gov (United States)

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  5. Electrochemical Detection of a Dengue-related Oligonucleotide Sequence Using Ferrocenium as a Hybridization Indicator

    OpenAIRE

    José Luiz de Lima-Filho; Duarte Miguel França dos Prazeres; ernando Rodrigues Ribeiro Teles

    2007-01-01

    A simple method for electrochemical detection of a synthetic 20-bp oligonucleotide sequence related with dengue virus genome was developed. A complimentary DNA probe sequence was electrostatically immobilized onto a glassy carbon electrode modified with chitosan. Electrochemical detection of hybridization between probe and target was performed by cyclic voltammetry, using ferrocene (Fc+) as a hybridization label. After hybridization, the peak current response of Fc+ oxidation increased around...

  6. MagiProbe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator

    OpenAIRE

    Yamane, Akio

    2002-01-01

    Fluorescence is the favored signaling technology for molecular diagnoses. Fluorescence energy transfer-based methods are powerful homogeneous assay tools. A novel oligonucleotide probe, named MagiProbe, which is simple to use, is described, and information given about the duplex formed with a target. The probe internally has a fluorophore and an intercalator. Its fluorescence is quenched by the intercalator in the absence of a target sequence. On hybridization with a target sequence, the prob...

  7. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

    NARCIS (Netherlands)

    Jansen, GJ; Mooibroek, M; Idema, J; Harmsen, HJM; Welling, GW; Degener, JE

    2000-01-01

    The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial spe

  8. Efficient oligonucleotide probe selection for pan-genomic tiling arrays

    Directory of Open Access Journals (Sweden)

    Zhang Wei

    2009-09-01

    Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

  9. The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

    Science.gov (United States)

    2013-01-01

    Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

  10. 'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

    Science.gov (United States)

    Mladinic, M; Didelon, F; Cherubini, E; Bradbury, A

    2000-05-15

    As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

  11. Fluorescent hybridization probes for nucleic acid detection.

    Science.gov (United States)

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  12. Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Mirza, M S; Rademaker, J L; Janse, J D; Akkermans, A D

    1993-11-01

    In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 degrees C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 degrees C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 degrees C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.

  13. ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

    Science.gov (United States)

    Stenberg, Johan; Nilsson, Mats; Landegren, Ulf

    2005-01-01

    Background Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. Results ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. Conclusion ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms. PMID:16171527

  14. probeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.

    Science.gov (United States)

    Greuter, Daniel; Loy, Alexander; Horn, Matthias; Rattei, Thomas

    2016-01-04

    probeBase http://www.probebase.net is a manually maintained and curated database of rRNA-targeted oligonucleotide probes and primers. Contextual information and multiple options for evaluating in silico hybridization performance against the most recent rRNA sequence databases are provided for each oligonucleotide entry, which makes probeBase an important and frequently used resource for microbiology research and diagnostics. Here we present a major update of probeBase, which was last featured in the NAR Database Issue 2007. This update describes a complete remodeling of the database architecture and environment to accommodate computationally efficient access. Improved search functions, sequence match tools and data output now extend the opportunities for finding suitable hierarchical probe sets that target an organism or taxon at different taxonomic levels. To facilitate the identification of complementary probe sets for organisms represented by short rRNA sequence reads generated by amplicon sequencing or metagenomic analysis with next generation sequencing technologies such as Illumina and IonTorrent, we introduce a novel tool that recovers surrogate near full-length rRNA sequences for short query sequences and finds matching oligonucleotides in probeBase.

  15. Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

    Science.gov (United States)

    Sorokin, N V; Chechetkin, V R; Pan'kov, S V; Somova, O G; Livshits, M A; Donnikov, M Y; Turygin, A Y; Barsky, V E; Zasedatelev, A S

    2006-08-01

    The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher

  16. chipD: a web tool to design oligonucleotide probes for high-density tiling arrays

    Science.gov (United States)

    Dufour, Yann S.; Wesenberg, Gary E.; Tritt, Andrew J.; Glasner, Jeremy D.; Perna, Nicole T.; Mitchell, Julie C.; Donohue, Timothy J.

    2010-01-01

    chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/. PMID:20529880

  17. Synthesis and hybridization properties of inverse oligonucleotides.

    OpenAIRE

    Marangoni, M.; Van Aerschot, Arthur; Augustijns, Patrick; Rozenski, Jef; Herdewijn , Piet

    1997-01-01

    The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and D...

  18. The use of oligonucleotide probes for meningococcal serotype characterization

    Directory of Open Access Journals (Sweden)

    SACCHI Claudio Tavares

    1998-01-01

    Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

  19. A spatially resolved nucleic acid biochip based on a gradient of density of immobilized probe oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sung Han [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6 (Canada); Krull, Ulrich [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6 (Canada)]. E-mail: ukrull@utm.utoronto.ca

    2006-04-06

    The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium-tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy{sub 3} and Cy{sub 5} dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0-200 ng/mm{sup 2}. The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions.

  20. ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

    Directory of Open Access Journals (Sweden)

    Nilsson Mats

    2005-09-01

    Full Text Available Abstract Background Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. Results ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. Conclusion ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms.

  1. Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

    Directory of Open Access Journals (Sweden)

    Jee Justin

    2008-12-01

    Full Text Available Abstract Background Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on S. cerevisiae and H. sapiens genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions. Results We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa. This skew is small in highly-expressed regions (± 0.5% of total intensity range and large (± 2% or more elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM. Conclusion We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.

  2. Experimental analysis of oligonucleotide microarray design criteria to detect deletions by comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Moerman Donald G

    2008-10-01

    Full Text Available Abstract Background Microarray comparative genomic hybridization (CGH is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. Results We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10°C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data

  3. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization.

    Science.gov (United States)

    Olschläger, Stephan; Günther, Stephan

    2012-07-01

    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

  4. Design and verification of a pangenome microarray oligonucleotide probe set for Dehalococcoides spp.

    Science.gov (United States)

    Hug, Laura A; Salehi, Maryam; Nuin, Paulo; Tillier, Elisabeth R; Edwards, Elizabeth A

    2011-08-01

    Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.

  5. Inference of subgenomic origin of BACs in an interspecific hybrid sugarcane cultivar by overlapping oligonucleotide hybridizations.

    Science.gov (United States)

    Kim, Changsoo; Robertson, Jon S; Paterson, Andrew H

    2011-09-01

    Sugarcane (Saccharum spp.) breeders in the early 20th century made remarkable progress in increasing yield and disease resistance by crossing Saccharum spontaneum L., a wild relative, to Saccharum officinarum L., a traditional cultivar. Modern sugarcane cultivars have approximately 71%-83% of their chromosomes originating from S. officinarum, approximately 10%-21% from S. spontaneum, and approximately 2%-13% recombinant or translocated chromosomes. In the present work, C(0)t-based cloning and sequencing (CBCS) was implemented to further explore highly repetitive DNA and to seek species-specific repeated DNA in both S. officinarum and S. spontaneum. For putatively species-specific sequences, overlappping oligonucleotide probes (overgos) were designed and hybridized to BAC filters from the interspecific hybrid sugarcane cultivar 'R570' to try to deduce parental origins of BAC clones. We inferred that 12 967 BACs putatively originated from S. officinarum and 5117 BACs from S. spontaneum. Another 1103 BACs were hybridized by both species-specific overgos, too many to account for by conventional recombination, thus suggesting ectopic recombination and (or) translocation of DNA elements. Constructing a low C(0)t library is useful to collect highly repeated DNA sequences and to search for potentially species-specific molecular markers, especially among recently diverged species. Even in the absence of repeat families that are species-specific in their entirety, the identification of localized variations within consensus sequences, coupled with the site specificity of short synthetic overgos, permits researchers to monitor species-specific or species-enriched variants.

  6. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This incre......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues....... This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within...

  7. Label-free detection of hybridization of oligonucleotides by oblique-incidence reflectivity difference method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.

  8. An anchored framework BAC map of mouse chromosome 11 assembled using multiplex oligonucleotide hybridization.

    Science.gov (United States)

    Cai, W W; Reneker, J; Chow, C W; Vaishnav, M; Bradley, A

    1998-12-15

    Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an "overgo" computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.

  9. Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

    Directory of Open Access Journals (Sweden)

    Ping Gong

    Full Text Available High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs. Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59% probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1 does not require a major genomics core facility; (2 allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3 is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4 if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is

  10. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    Science.gov (United States)

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  11. Environment-responsive fluorescent nucleoside analogue probe for studying oligonucleotide dynamics in a model cell-like compartment.

    Science.gov (United States)

    Pawar, Maroti G; Srivatsan, Seergazhi G

    2013-11-21

    The majority of fluorescent nucleoside analogue probes that have been used in the in vitro study of nucleic acids are not suitable for cell-based biophysical assays because they exhibit excitation maxima in the UV region and low quantum yields within oligonucleotides. Therefore, we propose that the photophysical characterization of oligonucleotides labeled with a fluorescent nucleoside analogue in reverse micelles (RM), which are good biological membrane models and UV-transparent, could provide an alternative approach to studying the properties of nucleic acids in a cell-like confined environment. In this context, we describe the photophysical properties of an environment-sensitive fluorescent uridine analogue (1), based on the 5-(benzo[b]thiophen-2-yl)pyrimidine core, in micelles and RM. The emissive nucleoside, which is polarity- and viscosity-sensitive, reports the environment of the surfactant assemblies via changes in its fluorescence properties. The nucleoside analogue, incorporated into an RNA oligonucleotide and hybridized to its complementary DNA and RNA oligonucleotides, exhibits a significantly higher fluorescence intensity, lifetime, and anisotropy in RM than in aqueous buffer, which is consistent with the environment of RM. Collectively, our results demonstrate that nucleoside 1 could be utilized as a fluorescent label to study the function of nucleic acids in a model cellular milieu.

  12. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

    Science.gov (United States)

    Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

    2015-02-07

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  13. Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

    Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

  14. Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

    Science.gov (United States)

    Hoehlig, Kai; Bethge, Lucas; Klussmann, Sven

    2015-01-01

    A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications. PMID:25679211

  15. 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.

  16. RNA probes, transcribed from synthetic DNA, for in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Brysch, W.; Hagendorff, G.; Schlingensiepen, K.H.

    1988-03-25

    Single stranded cRNA probes are ideal for in-situ-hybridization. Synthetic oligodesoxy-ribonucleotides on the other hand allow one to chose nucleotide sequences independently of restriction sites and availability of cloned templates. To combine the advantages of these two methods, the authors used an oligonucleotide, containing a T7-RNA-polymerase promotor sequence and a starting sequence of 6 bases as a template for an in-vitro-transcription reaction with T7-RNA-polymerase. A second oligonucleotide, complementary to basepairs 1-101 was also synthesized and the two strands heated to 95/sup 0/ for 3 min, then kept at 65/sup 0/C for one hour in 80 mM Tris, 12mM MgCl, 4 mM Spermidine, 0,04% Triton and finally cooled on ice. The resulting double stranded DNA was used as a template to transcribe /sup 35/S-labelled cRNA, using DNA, T7-Polymerase, /sup 35/S-UTP, ATP, GTP and CTP and RNasin (Promega). No difference could be observed comparing the resulting hybridization pattern with that obtained by using a plasmid derived cRNA probe of rat brain sodium channel II. Moreover the hybridization signal was clearly distinct from the background labelling resulting from hybridization with a sense control probe of the same specific activity.

  17. Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

    Science.gov (United States)

    Hakala, H; Virta, P; Salo, H; Lönnberg, H

    1998-12-15

    Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.

  18. Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    Science.gov (United States)

    Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

    2015-09-01

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

  19. Typing for HLA-DPB1*03 and HLA-DPB1*06 using allele-specific DNA in vitro amplification and allele-specific oligonucleotide probes. Detection of "new" DPB1*06 variants

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P;

    1989-01-01

    DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes has recently been reported. The resulting DNA amplification was specific for the HLA-DPB locus. Typing for the individual DPB alleles was exclusively dependent on the hybridizations of the probe...

  20. Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Proudnikov, D.; Kirillov, E.; Chumakov, K.; Donion, J.; Rezapkin, G.; Mirzabekov, A.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Center for Biologics Evaluation and Research

    2000-01-01

    This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

  1. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This incre...... the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples....

  2. Hybridization probe for femtomolar quantification of selected nucleic acid sequences on a disposable electrode.

    Science.gov (United States)

    Jenkins, Daniel M; Chami, Bilal; Kreuzer, Matthias; Presting, Gernot; Alvarez, Anne M; Liaw, Bor Yann

    2006-04-01

    Mixed monolayers of electroactive hybridization probes on gold surfaces of a disposable electrode were investigated as a technology for simple, sensitive, selective, and rapid gene identification. Hybridization to the ferrocene-labeled hairpin probes reproducibly diminished cyclic redox currents, presumably due to a displacement of the label from the electrode. Observed peak current densities were roughly 1000x greater than those observed in previous studies, such that results could easily be interpreted without the use of algorithms to correct for background polarization currents. Probes were sensitive to hybridization with a number of oligonucleotide sequences with varying homology, but target oligonucleotides could be distinguished from competing nontarget sequences based on unique "melting" profiles from the probe. Detection limits were demonstrated down to nearly 100 fM, which may be low enough to identify certain genetic conditions or infections without amplification. This technology has rich potential for use in field devices for gene identification as well as in gene microarrays.

  3. Complexes of carbon nanotubes with oligonucleotides in thin Langmuir-Blodgett films to detect electrochemically hybridization

    Science.gov (United States)

    Egorov, A. S.; Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Orekhovskaya, T. I.; Veligura, A. A.; Govorov, M. I.; Shulitsky, B. G.

    2014-10-01

    Self-assembled complexes consisting of thin multi-walled carbon nanotubes (MWCNTs) and DNA-oligonucleotides which are able to a cooperative binding to complementary oligonucleotides have been investigated. It was establised a high-performance charge transport in nanostructured Langmuir-Blodgett complexes thin MWCNTs/DNA. A method to electrochemically detect DNA hybridization on the self-organized structures has been proposed.

  4. A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Fairbanks Benjamin D

    2006-04-01

    Full Text Available Abstract Background DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. Results We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, β-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. Conclusion Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that

  5. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte Stahl; Jensen, Knud Jørgen

    2015-01-01

    results by electronic structure calculations. Functionalized oligonucleotides were prepared in good yields by protein-mediated CuAAC click reactions for the first time with a human copper-binding chaperon. The carbohydrate, peptide, and fluorescent derivatives display high binding affinity and selectivity...... 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...

  6. PTPan--overcoming memory limitations in oligonucleotide string matching for primer/probe design.

    Science.gov (United States)

    Eissler, Tilo; Hodges, Christopher P; Meier, Harald

    2011-10-15

    Nucleic acid diagnostics has high demands for non-heuristic exact and approximate oligonucleotide string matching concerning in silico primer/probe design in huge nucleic acid sequence collections. Unfortunately, public sequence repositories grow much faster than computer hardware performance and main memory capacity do. This growth imposes severe problems on existing oligonucleotide primer/probe design applications necessitating new approaches based on space-efficient indexing structures. We developed PTPan (spoken Peter Pan, 'PT' is for Position Tree, the earlier name of suffix trees), a space-efficient indexing structure for approximate oligonucleotide string matching in nucleic acid sequence data. Based on suffix trees, it combines partitioning, truncation and a new suffix tree stream compression to deal with large amounts of aligned and unaligned data. PTPan operates efficiently in main memory and on secondary storage, balancing between memory consumption and runtime during construction and application. Based on PTPan, applications supporting similarity search and primer/probe design have been implemented, namely FindFamily, ProbeMatch and ProbeDesign. All three use a weighted Levenshtein distance metric for approximative queries to find and rate matches with indels as well as substitutions. We integrated PTPan in the worldwide used software package ARB to demonstrate usability and performance. Comparing PTPan and the original ARB index for the very large ssu-rRNA database SILVA, we recognized a shorter construction time, extended functionality and dramatically reduced memory requirements at the price of expanded, but very reasonable query times. PTPan enables indexing of huge nucleic acid sequence collections at reasonable application response times. Not being limited by main memory, PTPan constitutes a major advancement regarding rapid oligonucleotide string matching in primer/probe design now and in the future facing the enormous growth of molecular

  7. DNA sequence analysis by hybridization with oligonucleotide microchips : MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Stomakhin, A. A.; Vasiliskov, V. A.; Timofeev, E.; Schulga, D.; Cotter, R. J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Moscow Inst. of Physics and Technology; Middle Atlantic Mass Spectrometry Lab.; Johns Hopkins Univ. School of Medicine

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15{sup o}C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

  8. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

    Science.gov (United States)

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale

    2014-02-01

    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  9. Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.

    OpenAIRE

    Torczynski, R M; Fuke, M; Bollon, A P

    1984-01-01

    A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence d...

  10. Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides.

    Science.gov (United States)

    Deo, Monika; Yu, Jenn-Yah; Chung, Kwan-Ho; Tippens, Melissa; Turner, David L

    2006-09-01

    We have developed an in situ hybridization procedure for the detection of microRNAs (miRNAs) in tissue sections from mouse embryos and adult organs. The method uses highly specific washing conditions for RNA oligonucleotide probes conjugated to a fluorescein hapten. We show that this method detects predominantly mature miRNAs rather than the miRNA precursors or primary transcripts. We have determined expression patterns for several miRNAs expressed in the developing and adult nervous system, including miR-124a, miR-9, miR-92, and miR-204. Whereas miR-124a is expressed in neurons, miR-9 is expressed in neural progenitors and some neurons, and miR-204 is expressed in the choroid plexus, retinal pigment epithelium, and ciliary body. miR-204 is located in an intron of the TRPM3 gene, and the TRPM3 mRNA is coexpressed with miR-204 in the choroid plexus. We also find that primary transcripts for miR-124a and miR-9 genes are expressed in patterns similar to their respective mature miRNAs. The ability to visualize expression of specific miRNAs in embryos and tissues should aid studies on miRNA function. Copyright 2006 Wiley-Liss, Inc.

  11. Evaluation of a fluorescence-labelled oligonucleotide tide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Christensen, H.; Wegener, Henrik Caspar

    1997-01-01

    -embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide...

  12. Fluorescence detection of natural RNA using rationally designed "clickable" oligonucleotide probes

    DEFF Research Database (Denmark)

    Okholm, Anders; Kjems, Jørgen; Astakhova, Kira

    2014-01-01

    Herein a reliable approach to the design of effective fluorescent probes for RNA detection is described. The fluorescence signalling of hybridization by internally positioned polyaromatic hydrocarbons and rhodamine dyes was achieved with a low fluorescence background signal, high fluorescence...

  13. Functionalization of magnetic gold/iron-oxide composite nanoparticles with oligonucleotides and magnetic separation of specific target

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Takuya [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)]. E-mail: t-kinoshita@mit.eng.osaka-u.ac.jp; Seino, Satoshi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Mizukoshi, Yoshiteru [Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Nakagawa, Takashi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamamoto, Takao A. [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2007-04-15

    Magnetic composite nanoparticles of gold and iron-oxide synthesized with gamma-rays or ultrasonics were functionalized with thiol-modified oligonucleotides. The amount of oligonucleotides bound to the functionalized nanoparticle probes via hybridization was quantified with fluorescently-labeled target oligonucleotides. Our composite nanoparticles magnetically separated the specific target oligonucleotides without the non-specific adsorption.

  14. Application of DNA fingerprinting with digoxigenated oligonucleotide probe (CAC)5 to analysis of the genetic variation within Taenia taeniaeformis.

    Science.gov (United States)

    Okamoto, M; Ueda, H; Hayashi, M; Oku, Y; Kurosawa, T; Kamiya, M

    1995-04-01

    DNA from T. taeniaeformis digested with the restriction endonuclease was hybridized with digoxigenated oligonucleotide probe (CAC)5. Metacestode and adult showed same clear multibanding patterns, which were characteristic of multilocus DNA fingerprinting. The fingerprinting patterns were quite different from those of the rodent hosts. Genetic variations in 4 laboratory-reared isolates of T. taeniaeformis, including 3 isolates which have been reported to be indistinguishable by infectivity, morphology and protein composition of metacestode, were investigated using this technique. Each of the 4 isolates exhibited isolate-specific fingerprinting patterns and were easily distinguished from one another, thus it was considered that (CAC)5 was a highly resolvable and informative probe for cestodes. However, it was also indicated that (CAC)5 was so sensitive that applying fingerprinting with (CAC)5 to taxonomical or phylogenetic analysis was limited where habitat of the host was restricted to the small area. In comparison to fingerprinting with 32P-labeled (CAC)5, fingerprinting with digoxigenated (CAC)5 represented more and sharper bands. It was considered that a digoxigenated probe was more useful for genetic analysis of cestodes.

  15. Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria.

    Science.gov (United States)

    Blasco, Lucía; Ferrer, Sergi; Pardo, Isabel

    2003-08-08

    A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).

  16. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  17. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  18. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    Institute of Scientific and Technical Information of China (English)

    Lian-Qun Jin; Jun-Wen Li; Sheng-Qi Wang; Fu-Huan Chao; Xin-Wei Wang; Zheng-Quan Yuan

    2005-01-01

    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately.METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays.RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified.CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus,Staphylococcus aureus, Proteus sp., Bacillus cereus,Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range,and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost.

  19. Design Considerations for Array CGH to OligonucleotideArrays

    Energy Technology Data Exchange (ETDEWEB)

    Baldocchi, R.A.; Glynne, R.J.; Chin, K.; Kowbel, D.; Collins, C.; Mack, D.H.; Gray, J.W.

    2005-03-04

    Background: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. Methods: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. Results: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. Conclusion: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

  20. Near-infrared silver cluster optically signaling oligonucleotide hybridization and assembling two DNA hosts.

    Science.gov (United States)

    Petty, Jeffrey T; Nicholson, David A; Sergev, Orlin O; Graham, Stuart K

    2014-09-16

    Silver clusters with ~10 atoms form within DNA strands, and the conjugates are chemical sensors. The DNA host hybridizes with short oligonucleotides, and the cluster moieties optically respond to these analytes. Our studies focus on how the cluster adducts perturb the structure of their DNA hosts. Our sensor is comprised of an oligonucleotide with two components: a 5'-cluster domain that complexes silver clusters and a 3'-recognition site that hybridizes with a target oligonucleotide. The single-stranded sensor encapsulates an ~11 silver atom cluster with violet absorption at 400 nm and with minimal emission. The recognition site hybridizes with complementary oligonucleotides, and the violet cluster converts to an emissive near-infrared cluster with absorption at 730 nm. Our key finding is that the near-infrared cluster coordinates two of its hybridized hosts. The resulting tertiary structure was investigated using intermolecular and intramolecular variants of the same dimer. The intermolecular dimer assembles in concentrated (~5 μM) DNA solutions. Strand stoichiometries and orientations were chromatographically determined using thymine-modified complements that increase the overall conjugate size. The intramolecular dimer develops within a DNA scaffold that is founded on three linked duplexes. The high local cluster concentrations and relative strand arrangements again favor the antiparallel dimer for the near-infrared cluster. When the two monomeric DNA/violet cluster conjugates transform to one dimeric DNA/near-infrared conjugate, the DNA strands accumulate silver. We propose that these correlated changes in DNA structure and silver stoichiometry underlie the violet to near-infrared cluster transformation.

  1. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: quantitation and optimization of a sandwich type assay.

    Science.gov (United States)

    Hakala, H; Mäki, E; Lönnberg, H

    1998-01-01

    Uniformly sized (50 micro m) porous glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as the solid phase in a sandwich type mixed-phase hybridization assay based on time-resolved fluorescence detection on a single particle. These particles were coated with oligodeoxyribonucleotide probes by conventional phosphoramidite chain assembly. An oligodeoxyribonucleotide bearing a photoluminescent europium(III) chelate, ¿2,2',2",2"'-¿¿4'-¿4"'-[(4, 6-dichloro-1,3,5-triazin-2-yl)amino]phenyl¿-2,2':6',2"-terpyrid ine-6, 6"-diyl¿bis(methylenenitrilo)¿tetrakis(acetato)¿eur opi um(III), was hybridized to a complementary sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound probes. The latter binding was quantified by time-resolved measurement of the emission signal of a single particle. Kinetics of hybridization and the effect of the concentration of the target oligomer and the fluorescently tagged probe on the efficiency of hybridization were studied. The intensity of the emission signal was linearly related to the concentration of the target oligomer over a range of 5 orders of magnitude. The length of the complementary region between the target oligomer and the particle-bound probe was varied, and the effect of point mutations and deletions on the hybridization efficiency was determined in each case. The maximal selectivity was observed with 10-16-base pair complementary sequences, the optimal length depending on the oligonucleotide loading on the particle. Discrimination between the complete matches and point mismatches was unequivocal, a single point mutation and/or deletion decreasing the efficiency of hybridization by more than 2 orders of magnitude.

  2. An algorithm and program for finding sequence specific oligo-nucleotide probes for species identification

    Directory of Open Access Journals (Sweden)

    Tautz Diethard

    2002-03-01

    Full Text Available Abstract Background The identification of species or species groups with specific oligo-nucleotides as molecular signatures is becoming increasingly popular for bacterial samples. However, it shows also great promise for other small organisms that are taxonomically difficult to tract. Results We have devised here an algorithm that aims to find the optimal probes for any given set of sequences. The program requires only a crude alignment of these sequences as input and is optimized for performance to deal also with very large datasets. The algorithm is designed such that the position of mismatches in the probes influences the selection and makes provision of single nucleotide outloops. Program implementations are available for Linux and Windows.

  3. Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

    DEFF Research Database (Denmark)

    Barken, K.B.; Cabig-Ciminska, M.; Holmgren, A.;

    2004-01-01

    Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding sit....... Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.......Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site...

  4. A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes.

    Science.gov (United States)

    Wyroba, E; Satir, B H

    2000-01-01

    Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.

  5. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    Science.gov (United States)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    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 attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

  6. Photoswitchable oligonucleotide-modified gold nanoparticles: controlling hybridization stringency with photon dose.

    Science.gov (United States)

    Yan, Yunqi; Chen, Jennifer I L; Ginger, David S

    2012-05-09

    We describe a new class of stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic directed assembly and sensing behaviors associated with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces a trans-cis isomerization of the azobenzene which destabilizes the DNA duplex, resulting in dissociation of the nanoparticle assemblies. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and we demonstrate that photon dose can thus be used in place of temperature or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.

  7. Reactive Microcontact Printing of DNA Probes on (DMA-NAS-MAPS) Copolymer-Coated Substrates for Efficient Hybridization Platforms.

    Science.gov (United States)

    Castagna, Rossella; Bertucci, Alessandro; Prasetyanto, Eko Adi; Monticelli, Marco; Conca, Dario Valter; Massetti, Matteo; Sharma, Parikshit Pratim; Damin, Francesco; Chiari, Marcella; De Cola, Luisa; Bertacco, Riccardo

    2016-04-05

    High-performing hybridization platforms fabricated by reactive microcontact printing of DNA probes are presented. Multishaped PDMS molds are used to covalently bind oligonucleotides over a functional copolymer (DMA-NAS-MAPS) surface. Printed structures with minimum width of about 1.5 μm, spaced by 10 μm, are demonstrated, with edge corrugation lower than 300 nm. The quantification of the immobilized surface probes via fluorescence imaging gives a remarkable concentration of 3.3 × 10(3) oligonucleotides/μm(2), almost totally active when used as probes in DNA-DNA hybridization assays. Indeed, fluorescence and atomic force microscopy show a 95% efficiency in target binding and uniform DNA hybridization over printed areas.

  8. Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

    Science.gov (United States)

    Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

    2016-01-15

    Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors.

  9. SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies

    Science.gov (United States)

    Matsishin, M. J.; Ushenin, Iu. V.; Rachkov, A. E.; Solatkin, A. P.

    2016-01-01

    In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

  10. Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Aznar, R; Ludwig, W; Amann, R I; Schleifer, K H

    1994-04-01

    A comparative analysis of seven new 16S rRNA gene sequences of pathogenic Vibrio species with previously published vibrio sequences confirmed that Vibrio vulnificus represents a group that is not closely related to the core organisms of the genus Vibrio. In addition, we found that V. vulnificus, Listonella (Vibrio) anguillarum and Vibrio diazotrophicus branch off separately from the core group. A comparison of the 16S rRNA gene sequences of V. vulnificus strains belonging to biotypes 1 and 2 revealed that the sequences of all but four biotype 1 strains were identical to each other but slightly different (17 bases) from the sequences of the rest of the V. vulnificus strains investigated. In addition, the sequences of variable regions of the 23S rRNA genes of Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibrio cholerae, and V. vulnificus C7184 and TW1 were determined, aligned, and compared with all available bacterial 23S rRNA sequences in order to search for specific target sites. As a result, four oligonucleotide probes specific for V. vulnificus were synthesized, and the specificities of these probes were evaluated by dot blot hybridization to membrane-bound RNAs from 21 V. vulnificus strains, 13 strains belonging to other Vibrio species, 61 strains belonging to species that are members of the alpha, beta, and gamma subclasses of the Proteobacteria, and 3 eucaryotic microorganisms. Two probes hybridized with all of the V. vulnificus strains tested, and the other two probes distinguished V. vulnificus biotype 1 strains from all other organisms. In situ identification of V. vulnificus by using tetramethylrhodamine- or fluorescein-labelled oligonucleotides is now possible.

  11. Effect of LNA- and OMeN-modified oligonucleotide probes on the stability and discrimination of mismatched base pairs of duplexes

    Indian Academy of Sciences (India)

    Ying Yan; Jing Yan; Xianyu Piao; Tianbiao Zhang; Yifu Guan

    2012-06-01

    Locked nucleic acid (LNA) and 2′--methyl nucleotide (OMeN) are the most extensively studied nucleotide analogues. Although both LNA and OMeN are characterized by the C3′-endo sugar pucker conformation, which is dominant in A-form DNA and RNA nucleotides, they demonstrate different binding behaviours. Previous studies have focused attention on their properties of duplex stabilities, hybridization kinetics and resistance against nuclease digestion; however, their ability to discriminate mismatched hybridizations has been explored much less. In this study, LNA- and OMeN-modified oligonucleotide probes have been prepared and their effects on the DNA duplex stability have been examined: LNA modifications can enhance the duplex stability, whereas OMeN modifications reduce the duplex stability. Next, we studied how the LNA:DNA and OMeN:DNA mismatches reduced the duplex stability. Melting temperature measurement showed that different LNA:DNA or OMeN:DNA mismatches indeed influence the duplex stability differently. LNA purines can discriminate LNA:DNA mismatches more effectively than LNA pyrimidines as well as DNA nucleotides. Furthermore, we designed five LNA- and five OMeN-modified oligonucleotide probes to simulate realistic situations where target–probe duplexes contain a complementary LNA:DNA or OMeN:DNA base pairs and a DNA:DNA mismatch simultaneously. The measured collective effect showed that the duplex stability was enhanced by the complementary LNA:DNA base pair but decreased by the DNA:DNA mismatch in a position-dependent manner regardless of the chemical identity and position of the complementary LNA:DNA base pair. On the other hand, the OMeN-modified probes also showed that the duplex stability was reduced by both the OMeN modification and the OMeN:DNA mismatch in a position-dependent manner.

  12. Radiolabeled oligonucleotides for antisense imaging

    Science.gov (United States)

    Iyer, Arun K; He, Jiang

    2011-01-01

    Oligonucleotides radiolabeled with isotopes emitting γ-rays (for SPECT imaging) or positrons (for PET imaging) can be useful for targeting messenger RNA (mRNA) thereby serving as non-invasive imaging tools for detection of gene expression in vivo (antisense imaging). Radiolabeled oligonucleotides may also be used for monitoring their in vivo fate, thereby helping us better understand the barriers to its delivery for antisense targeting. These developments have led to a new area of molecular imaging and targeting, utilizing radiolabeled antisense oligonucleotides. However, the success of antisense imaging relies heavily on overcoming the barriers for its targeted delivery in vivo. Furthermore, the low ability of the radiolabeled antisense oligonucleotide to subsequently internalize into the cell and hybridize with its target mRNA poses additional challenges in realizing its potentials. This review covers the advances in the antisense imaging probe development for PET and SPECT, with an emphasis on radiolabeling strategies, stability, delivery and in vivo targeting. PMID:21822406

  13. Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities

    Directory of Open Access Journals (Sweden)

    Bathe Stephan

    2006-06-01

    Full Text Available Abstract Background The β-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. Results We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. Conclusion The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

  14. Changes in the end-to-end distance distribution in an oligonucleotide following hybridization

    Science.gov (United States)

    Parkhurst, Lawrence J.; Parkhurst, Kay M.

    1994-08-01

    A 16-mer deoxy oligonucleotide was labeled at the 5' end with x- rhodamine and at the 3' end with fluorescein. The fluorescence lifetime of the donor, fluorescein, under conditions for resonance energy transfer, was studied using the SLM 4850 multiharmonic frequency phase fluorometer in order to obtain information on the end-to-end distance distribution P(R) in the oligomer. When this doubly labeled oligonucleotide was hybridized to its 16-mer complement, the fluorescein fluorescence decay could be very well described by a P(R) that was a symmetric shifted Gaussian with center at 68.4 angstrom and (sigma) equals6.4 angstrom. Simulations suggested that part of the width might be attributable to a distribution in (kappa) 2. In the single- stranded labeled oligomer, there was enhanced energy transfer from the fluorescein to the rhodamine and the best fitting symmetrical shifted Gaussian representation of P(R) was centered at 53.8 angstrom with (kappa) equals6.9 angstrom. There was significant lack of fit with this model, however. A model independent procedure was developed for extracting P(R) as a sum of weighted Hermite polynomials. This procedure gave a P(R) with a large negative region at R<20 angstrom, suggesting that rotational averaging for (kappa) 2 was not quite complete prior to significant decay of the donor excited state.

  15. Genomic analysis by oligonucleotide array Comparative Genomic Hybridization utilizing formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Savage, Stephanie J; Hostetter, Galen

    2011-01-01

    Formalin fixation has been used to preserve tissues for more than a hundred years, and there are currently more than 300 million archival samples in the United States alone. The application of genomic protocols such as high-density oligonucleotide array Comparative Genomic Hybridization (aCGH) to formalin-fixed, paraffin-embedded (FFPE) tissues, therefore, opens an untapped resource of available tissues for research and facilitates utilization of existing clinical data in a research sample set. However, formalin fixation results in cross-linking of proteins and DNA, typically leading to such a significant degradation of DNA template that little is available for use in molecular applications. Here, we describe a protocol to circumvent formalin fixation artifact by utilizing enzymatic reactions to obtain quality DNA from a wide range of FFPE tissues for successful genome-wide discovery of gene dosage alterations in archival clinical samples.

  16. Aptamer-gelatin composite for a trigger release system mediated by oligonucleotide hybridization.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Srakaew, Prangkamol; Naramitpanich, Pajaree

    2014-01-01

    Nucleic acid aptamers not only specifically bind to their target proteins with high affinity but also form intermolecular hybridization with their complementary oligonucleotides (CO). The hybridization can interrupt aptamer/protein interaction due to the changes of aptamer secondary structure which rely on hybridization length and base-pairing positions. Herein we aim to use this unique property of the aptamers, when combined with gelatin to develop a novel composite with desirable protein release profiles. Platelet-derived growth factor-BB (PDGF-BB) and its aptamer were used as target molecules. Prior to performing the release study, the effects of CO on aptamer-protein interaction were observed by surface plasmon resonance (SPR). The SPR sensorgram indicated that the aptamer dissociated from the bounded proteins when it hybridized with the CO. The aptamer was then immobilized onto streptavidin coated polystyrene particles via biotin/streptavidin interaction. Then, PDGF-BB and aptamer functionalized particles were mixed with gelatin solution and cast as small pieces of composite. The success of the composite preparation was confirmed by flow cytometry and microscopy. PDGF-BB release at several time points was quantified by ELISA. The results showed that the aptamer-gelatin composite could slow the release rate of the proteins from the composite due to strong binding of proteins and aptamers. Once the CO was added to the system, the release rate was significantly enhanced because the aptamer hybridized with the CO and lost its active secondary structure. Therefore, the proteins were triggered to release out from the composite. This work suggests a promising strategy for controlling the release of bioactive molecules in medical treatments.

  17. Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining.

    Science.gov (United States)

    Cubie, H A; Norval, M

    1989-09-01

    Human papilloma virus was detected by in situ hybridisation in routinely processed paraffin wax sections using a synthetically produced oligonucleotide probe, end-labelled with biotin, and amplified with anti-biotin-immunogold silver staining (anti-biotin-IGSS). This system proved more sensitive than amplification with streptavidin-biotinylated alkaline phosphatase for detecting human papilloma virus type 16 in cervical tissues. The method was successfully combined with antigen staining for papilloma virus common antigens in skin and genital warts. This simple and quick method, using non-radioactively labelled synthetic probes, may be useful for the detection of other viruses in stored material and may be suitable for other double staining procedures.

  18. Probing Compositional Variation within Hybrid Nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Yuhas, Benjamin D.; Habas, Susan E.; Fakra, Sirine C.; Mokari, Taleb

    2010-06-22

    We present a detailed analysis of the structural and magnetic properties of solution-grown PtCo-CdS hybrid structures in comparison to similar free-standing PtCo alloy nanoparticles. X-ray absorption spectroscopy is utilized as a sensitive probe for identifying subtle differences in the structure of the hybrid materials. We found that the growth of bimetallic tips on a CdS nanorod substrate leads to a more complex nanoparticle structure composed of a PtCo alloy core and thin CoO shell. The core-shell architecture is an unexpected consequence of the different nanoparticle growth mechanism on the nanorod tip, as compared to free growth in solution. Magnetic measurements indicate that the PtCo-CdS hybrid structures are superparamagnetic despite the presence of a CoO shell. The use of X-ray spectroscopic techniques to detect minute differences in atomic structure and bonding in complex nanosystems makes it possible to better understand and predict catalytic or magnetic properties for nanoscale bimetallic hybrid materials.

  19. Hybridization kinetics analysis of an oligonucleotide microarray for microRNA detection

    Institute of Scientific and Technical Information of China (English)

    Botao Zhao; Shuo Ding; Wei Li; Youxin Jin

    2011-01-01

    MicroRNA (miRNA) microarrays have been successfully used for profiling miRNA expression in many physiological processes such as development, differentiation, oncogenesis,and other disease processes. Detecting miRNA by miRNA microarray is actually based on nucleic acid hybridization between target molecules and their corresponding complementary probes. Due to the small size and high degree of similarity among miRNA sequences, the hybridization condition must be carefully optimized to get specific and reliable signals. Previously, we reported a microarray platform to detect miRNA expression. In this study, we evaluated the sensitivity and specificity of our microarray platform. After systematic analysis, we determined an optimized hybridization condition with high sensitivity and specificity for miRNA detection. Our results would be helpful for other hybridization-based miRNA detection methods, such as northern blot and nuclease protection assay.

  20. Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

    Science.gov (United States)

    Cheglakov, Zoya

    Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide

  1. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    Science.gov (United States)

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).

  2. Mechanism of antisense oligonucleotide interaction with natural RNAs.

    Science.gov (United States)

    Serikov, R; Petyuk, V; Vorobijev, Y; Koval, V; Fedorova, O; Vlassov, V; Zenkova, M

    2011-08-01

    Oligonucleotides find several numbers of applications: as diagnostic probes, RT and PCR primers and antisense agents due to their ability of forming specific interactions with complementary nucleotide sequences within nucleic acids. These interactions are strongly affected by accessibility of the target sequence in the RNA structure. In the present work the mechanism of invasion of RNA structure by oligonucleotide was investigated using a model system: yeast tRNA(Phe) and oligonucleotides complementary to the 3'-part of this molecule. Kinetics of interaction of oligonucleotides with in vitro transcript of yeast tRNAPhe was studied using stopped-flow technique with fluorescence quenching detection, 5'-DABCYL labeled oligonucleotide was hybridized with 3'-fluorescein labeled tRNA(Phe). The results evidence for a four-step invasion process of the oligonucleotide-RNA complex formation. The process is initiated by formation of transition complexes with nucleotides in the T-loop and ACCA sequence. This complex formation is followed by RNA unfolding and formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Computer modeling of oligonucleotide-tRNA(Phe) interaction revealed potential factors that could favor transition complexes formation and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular "wedge". Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA-RNA interactions. The obtained results can be used for choosing efficient oligonucleotide probes.

  3. Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Awsiuk, K., E-mail: kamil.awsiuk@uj.edu.pl [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Rysz, J. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Petrou, P. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Budkowski, A. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Bernasik, A. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Kakabakos, S. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Marzec, M.M. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Raptis, I. [Institute for Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece)

    2014-01-30

    To immobilize effectively oligonucleotide probes on SiO{sub 2} modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m{sup 2}) and second (1.31(±0.22) mg/m{sup 2}) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m{sup 2}) and fourth (0.41(±0.11) mg/m{sup 2}) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  4. 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.

  5. Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package

    Directory of Open Access Journals (Sweden)

    Meier Harald

    2006-05-01

    Full Text Available Abstract Background Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment. Results Three-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA. Conclusion Visualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating

  6. Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

    Directory of Open Access Journals (Sweden)

    Mattiuz Pier

    2005-10-01

    Full Text Available Abstract Background The minor histocompatibility antigens (mHags are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.

  7. 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.

  8. 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

    2013-02-07

    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. SINGLE CELL DEGENERATE OLIGONUCLEOTIDE PRIMER-PCR AND COMPARATIVE GENOMIC HYBRIDIZATION WITH MODIFIED CONTROL REFERENCE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    For investigating the possibility of applying degenerate oligonucleotide primer PCR (DOP-PCR) and comparative genomic hybridization (CGH) technique to analyses of genomic genetics in a single cell, the whole genomic DNA of a single cell with XX, XY, XO, XXY, +13 or +21 was amplified by DOP-PCR. Single cell DOP-PCR CGHs with conventional and modified control references, the genomic DNA and a single cell DOP-PCR product from normal male, were carried out respectively. The results showed that the average profile of the fluorescence intensity ratio in CGH with the genomic DNA as reference fluctuates much and that the standard deviation in about 30% haploid is beyond the normal limits. False positive hyper-representation was found to exist in X chromosome while trisomy 13 and 21 were not detected. However, the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as reference were quite acceptable. The copy number changes of chromosome X,Y,13 and 21 were revealed. Those results suggested that there is unrandom unequal amplification in a single cell DOP-PCR. Using a single DOP-PCR product as reference can decrease its influence on CGH. Single cell DOP-PCR-CGH and its application in the genetic analyses of preimplantation embryo or fetal cell in maternal blood may be possible.

  10. Photoelectrochemical competitive DNA hybridization assay using semiconductor quantum dot conjugated oligonucleotides.

    Science.gov (United States)

    Baş, Deniz; Boyaci, Ismail Hakki

    2011-05-01

    A competitive DNA hybridization assay based on the photoelectrochemistry of the semiconductor quantum dot-single stranded DNA conjugates (QD-ssDNA) was developed. Hybridization of QD-ssDNA with the capture probe DNA immobilized on the indium-tin oxide electrodes enables photocurrent generation when the electrochemical cell was illuminated with a light source. Upon the competition between QD-ssDNA and single-stranded target DNA, the photocurrent response decreased with the increase in the target DNA concentration. A linear relationship between the photocurrent and the target DNA concentration was obtained (R(2) = 0.991). The selectivity of system towards the target DNA was also demonstrated using non-complementary sample.

  11. Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

    Directory of Open Access Journals (Sweden)

    Ivan B. Brukner

    2010-04-01

    Full Text Available This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18. These probes are obtained through in vitro selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109 and human DNA concentrations (10-100 ng as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

  12. Novel oligonucleotide probes for in situ detection of pederin-producing endosymbionts of Paederus riparius rove beetles (Coleoptera: Staphylinidae).

    Science.gov (United States)

    Kador, Matthias; Horn, Marcus A; Dettner, Konrad

    2011-06-01

    Bacterial endosymbionts from female Paederus rove beetles are hitherto uncultured, phylogenetically related to Pseudomonas sp., and produce the polyketide pederin, which exhibits strong cytotoxic effects and antitumoral activities. The location of such endosymbionts inside beetles and on beetles' eggs is hypothesized based on indirect evidence rather than elucidated. Thus, an endosymbiont-specific and a competitor oligonucleotide probe (Cy3-labelled PAE444 and unlabelled cPAE444, respectively) were designed and utilized for FISH with semi-thin sections of Paederus riparius eggs. Cy3-PAE444-positive cells were densely packed and covered the whole eggshell. Hundred percent of EUB338-Mix-positive total bacterial cells were PAE444 positive, indicating a biofilm dominated by Paederus endosymbionts. Analysis of different egg deposition stadiums by electron microscopy and pks (polyketide synthase gene, a structural gene associated with pederin biosynthesis)-PCR supported results obtained by FISH and revealed that the endosymbiont-containing layer is applied to the eggshell inside the efferent duct. These findings suggest that P. riparius endosymbionts are located inside unknown structures of the female genitalia, which allow for a well-regulated release of endosymbionts during oviposition. The novel oligonucleotide probes developed in this study will facilitate (1) the identification of symbiont-containing structures within genitalia of their beetle hosts and (2) directed cultivation approaches in the future.

  13. Fluorescence In Situ Hybridization Probe Preparation.

    Science.gov (United States)

    Tolomeo, Doron; Stanyon, Roscoe R; Rocchi, Mariano

    2017-01-01

    The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A "golden path" of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800-1000 bp) at each end allowed them to be very precisely mapped on the human genome.The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56-64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.

  14. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    Directory of Open Access Journals (Sweden)

    Thurnheer Thomas

    2011-01-01

    Full Text Available Abstract Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of

  15. Application of hybridization control probe to increase accuracy on ligation detection or minisequencing diagnostic microarrays

    Directory of Open Access Journals (Sweden)

    Hultman Jenni

    2009-12-01

    Full Text Available Abstract Background Nucleic acid detection based on ligation reaction or single nucleotide extension of ssDNA probes followed by tag microarray hybridization provides an accurate and sensitive detection tool for various diagnostic purposes. Since microarray quality is crucial for reliable detection, these methods can benefit from correcting for microarray artefacts using specifically adapted techniques. Findings Here we demonstrate the application of a per-spot hybridization control oligonucleotide probe and a novel way of computing normalization for tag array data. The method takes into account the absolute value of the detection probe signal and the variability in the control probe signal to significantly alleviate problems caused by artefacts and noise on low quality microarrays. Conclusions Diagnostic microarray platforms require experimental and computational tools to enable efficient correction of array artefacts. The techniques presented here improve the signal to noise ratio and help in determining true positives with better statistical significance and in allowing the use of arrays with poor quality that would otherwise be discarded.

  16. LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

    Directory of Open Access Journals (Sweden)

    Priya Natarajan

    2012-05-01

    Full Text Available Abstract Background Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH, is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. Results In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers while the other a secondary endosymbiont Arsenophonus (and present in less numbers. Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. Conclusion By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction

  17. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

    Science.gov (United States)

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C

    1999-11-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

  18. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotroph

  19. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotroph

  20. Single and multiple molecular beacon probes for DNA hybridization studies on a silica glass surface

    Science.gov (United States)

    Fang, Xiaohong; Liu, Xiaojing; Tan, Weihong

    1999-05-01

    Surface immobilizable molecular beacons have been developed for DNA hybridization studies on a silica glass plate. Molecular beacons are a new class of oligonucleotide probes that have a loop-and-stem structure with a fluorophore and a quencher attached to the two ends of the stem. They only emit intense fluorescence when hybridize to their target molecules. This provides an excellent selectivity for the detection of DNA molecules. We have designed biotinylated molecular beacons which can be immobilized onto a solid surface. The molecular beacon is synthesized using DABCYL as the quencher and an optical stable dye, tetramethylrhodamine, as the fluorophore. Mass spectrometry is used to confirm the synthesized molecular beacon. The molecular beacons have been immobilized onto a silica surface through biotin-avidin binding. The surface immobilized molecular beacons have been used for the detection of target DNA with subnanomolar analytical sensitivity. have also immobilized two different molecular beacons on a silica surface in spatially resolved microscopic regions. The hybridization study of these two different molecular beacon probes has shown excellent selectivity for their target sequences. The newly designed molecular beacons are intended for DNA molecular interaction studies at an interface and for the development of ultrasensitive DNA sensors for a variety of applications including disease diagnosis, disease mechanism studies, new drug development, and in the investigation of molecular interactions between DNA molecules and other interesting biomolecules.

  1. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    Science.gov (United States)

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-03-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.

  2. Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

    Science.gov (United States)

    Arantes, Thales Domingos; Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Bagagli, Eduardo

    2017-01-01

    BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis. PMID:28177048

  3. Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

    Energy Technology Data Exchange (ETDEWEB)

    Noor, M. Omair; Hrovat, David [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Moazami-Goudarzi, Maryam [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Espie, George S. [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada)

    2015-07-23

    Highlights: • Solid-phase QD-FRET transduction of isothermal tHDA amplicons on paper substrates. • Ratiometric QD-FRET transduction improves assay precision and lowers the detection limit. • Zeptomole detection limit by an iPad camera after isothermal amplification. • Tunable assay sensitivity by immobilizing different amounts of QD–probe bioconjugates. - Abstract: Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non

  4. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    microscopy and nucleic acid analogues have been proposed so far. METHODS AND RESULTS: Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

  5. Heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence of a rhodamine label at oxide-coated aluminum and silicon electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Spehar-Deleze, Anna-Maria [Laboratory of Sensors, Actuators and Microsystems, Institute of Microtechnology, University of Neuchatel, Rue Jaquet-Droz 1, CH-2007 Neuchatel (Switzerland) and Laboratory of Inorganic and Analytical Chemistry, Helsinki University of Technology, Kemistintie 1, FIN-02015 HUT (Finland)]. E-mail: anna-maria.spehar@unine.ch; Suomi, Johanna [Laboratory of Inorganic and Analytical Chemistry, Helsinki University of Technology, Kemistintie 1, FIN-02015 HUT (Finland); Jiang Qinghong [Laboratory of Inorganic and Analytical Chemistry, Helsinki University of Technology, Kemistintie 1, FIN-02015 HUT (Finland); Rooij, Nico de [Laboratory of Sensors, Actuators and Microsystems, Institute of Microtechnology, University of Neuchatel, Rue Jaquet-Droz 1, CH-2007 Neuchatel (Switzerland); Koudelka-Hep, Milena [Laboratory of Sensors, Actuators and Microsystems, Institute of Microtechnology, University of Neuchatel, Rue Jaquet-Droz 1, CH-2007 Neuchatel (Switzerland); Kulmala, Sakari [Laboratory of Inorganic and Analytical Chemistry, Helsinki University of Technology, Kemistintie 1, FIN-02015 HUT (Finland)

    2006-07-28

    This paper describes a heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence (HECL) of a rhodamine label. Thin oxide-film coated aluminum and silicon electrodes were modified with an aminosilane layer and derivatized with short, 15-mer oligonucleotides via diisothiocyanate coupling. Target oligonucleotides were conjugated with tetramethylrhodamine (TAMRA) dye at their amino modified 5' end and hybridization was detected using HECL of TAMRA. Preliminary results indicate sensitivity down to picomolar level and low nonspecific adsorption. The sensitivity was better on oxide-coated silicon compared to oxide-coated aluminum electrodes and two-base pair mismatched hybrids were successfully discriminated. The experimental results presented here might be useful for the design of disposable electrochemiluminescent DNA biosensors.

  6. Identification of rifampin-resistant mycobacterium tuberculosis strains by hybridization, PCR, and ligase detaction reaction on oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Mikhailovich, V.; Lapa, S.; Gryadunov, D.; Sobolev, A.; Strizhkov, B.; Chernyh, N.; Skotnikova, O.; Irtuganova, O.; Moroz, A.; Litvinov, V.; Vladimirskii, M.; Perelman, M.; Chernousova, L.; Erokhin, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences; Moscow Antituberculosis Center; Moscow Medical Academy; Russian Academy of Medical Sciences

    2001-07-01

    Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches -- hybridization, PCR, and ligase detection reaction -- were designed to analyze an 81-bp fragment of the gene rpoB encoding the {beta}-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

  7. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    Science.gov (United States)

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Exploring the Hybridization Thermodynamics of Spherical Nucleic Acids to Tailor Probes for Diagnostic and Therapeutic Applications

    Science.gov (United States)

    Randeria, Pratik Shailesh

    Spherical nucleic acids (SNAs), three-dimensional nanoparticle conjugates composed of densely packed and highly oriented oligonucleotides around organic or inorganic nanoparticles, are an emergent class of nanostructures that show promise as single-entity agents for intracellular messenger RNA (mRNA) detection and gene regulation. SNAs exhibit superior biocompatibility and biological properties compared to linear oligonucleotides, enabling them to overcome many of the limitations of linear oligonucleotides for use in biomedical applications. However, the origins of these biologically attractive properties are not well understood. In this dissertation, the chemistry underlying one such property is studied in detail, and the findings are applied towards the rational design of more effective SNAs for diagnostic and therapeutic applications. Chapter 1 introduces the synthesis of SNAs, the unique properties that make them superior to linear nucleic acids for biomedicine, and previously studied applications of these structures. Chapter 2 focuses on quantitatively studying the impact of the chemical structure of the SNA on its ability to hybridize multiple complementary nucleic acids. This chapter lays the groundwork for understanding the factors that govern SNA hybridization thermodynamics and how to tailor SNAs to increase their binding affinity to target mRNA strands. Chapters 3 and 4 capitalize on this knowledge to engineer probes for intracellular mRNA detection and gene regulation applications. Chapter 3 reports the development of an SNA-based probe that can simultaneously report the expression level of two different mRNA transcripts in live cells and differentiate diseased cells from non-diseased cells. Chapter 4 investigates the use of topically-applied SNAs to down-regulate a critical mediator of impaired wound healing in diabetic mice to accelerate wound closure. This study represents the first topical therapeutic application of SNA nanotechnology to treat open

  9. A signal-on electrochemical DNA biosensor based on potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition mediated labeling of hairpin-like oligonucleotide with electroactive probe.

    Science.gov (United States)

    Hu, Qiong; Kong, Jinming; Li, Yajie; Zhang, Xueji

    2016-01-15

    A novel electrochemical biosensor was developed for the signal-on detection of sequence-specific DNA by exploiting potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (φCuAAC) as an efficient approach for the labeling of hairpin-like oligonucleotide (hairpin) with electroactive probe. The hairpins, dually labeled with thiol and azide at either terminal, were firstly self-assembled on gold electrode and served as the capture probes for the specific recognition of target DNA. Upon hybridization with target DNA, the surface-confined hairpins were unfolded, liberating the azide-containing terminals away from electrode surface. Subsequently, the unfolded hairpins were conveniently and efficiently labeled with ethynylferrocene (EFC) via the φCuAAC. The quantitatively labeled EFC was finally measured via differential pulse voltammetry (DPV) for the signal-on electrochemical detection of sequence-specific DNA. The biosensor presented a good linear response over the range from 1pM to 1nM with a detection limit of 0.62pM. Results also revealed that it was highly specific and held a good detection capability in serum samples. Furthermore, the ability to chemoselectively label hairpin-like oligonucleotide with signal reporter by electrical addressing, together with the simplicity and efficiency of the φCuAAC, makes it compatible with microfluidic devices and microelectrode arrays to achieve the miniaturized and multiplexed detections.

  10. Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

    Science.gov (United States)

    Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

    2014-07-03

    Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

  11. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    Science.gov (United States)

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

    2016-10-01

    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. A novel fluorescent in situ hybridization technique for detection of Rickettsia spp. in archival samples

    DEFF Research Database (Denmark)

    Svendsen, Claus Bo; Boye, Mette; Struve, Carsten

    2009-01-01

    A novel, sensitive and specific method for detecting Rickettsia spp. in archival samples is described. The method involves the use of fluorescently marked oligonucleotide probes for in situ hybridization. Specific hybridization of Ricekttsia was found without problems of cross-reactions with bact......A novel, sensitive and specific method for detecting Rickettsia spp. in archival samples is described. The method involves the use of fluorescently marked oligonucleotide probes for in situ hybridization. Specific hybridization of Ricekttsia was found without problems of cross...

  13. 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.

  14. Improving signal intensities for genes with low-expression on oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hu Limei

    2004-06-01

    Full Text Available Abstract Background DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. Results Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. Conclusions We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays.

  15. Dendrimer-encapsulated copper as a novel oligonucleotides label for sensitive electrochemical stripping detection of DNA hybridization.

    Science.gov (United States)

    Gao, Huan; Jiang, Xue; Dong, Yang-Jun; Tang, Wan-Xin; Hou, Cong; Zhu, Ning-Ning

    2013-10-15

    This paper describes the synthesis and characterization of a novel electrochemical label for sensitive electrochemical stripping detection of DNA hybridization based on dendrimer-encapsulated copper. The generation 4.5 (G 4.5) carboxyl-terminated poly(amidoamine) dendrimer with a trimesyl core was used as a template for synthesis of Cu²⁺/dendrimer nanocomposites (Cu-DNCs). Ratios of Cu²⁺/dendrimer were optimized in order to obtain stable nanocomposites with maximal copper loading in the interior of a polymeric shell. Cu-DNCs labeled DNA probe was employed for determining a target ssDNA immobilized on multi-walled carbon nanotubes-modified glassy carbon electrode (GCE) based on a specific hybridization reaction. The hybridization events were monitored by electrochemical detection of Cu anchored on the hybrids after the release in a diluted nitric acid by anodic stripping differential pulse voltammetry (ASDPV). The results showed that only a complementary sequence could form a dsDNA with the Cu-DNCs DNA probe and give an obvious electrochemical signal. The non-complementary sequence exhibited negligible signal change compared with the blank measurement (means: the electrode containing no target DNA incubating in hybridization buffer solution containing Cu-DNCs DNA probe for a certain time). The use of Cu encapsulated-dendrimer as tags and ASDPV for the detection of the released Cu ions could enhance the hybridization signal, and result in the increase of the sensitivity for the target DNA. Under the conditions employed here, the detection limit for measuring the full complementary sequence is down to pM level. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Typing of enteroviruses by use of microwell oligonucleotide arrays.

    Science.gov (United States)

    Susi, P; Hattara, L; Waris, M; Luoma-Aho, T; Siitari, H; Hyypiä, T; Saviranta, P

    2009-06-01

    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

  17. Sequencing of megabase plus DNA by hybridization: Method development ENT. Final technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    Crkvenjakov, R.; Drmanac, R.

    1991-01-31

    Sequencing by hybridization (SBH) is the only sequencing method based on the experimental determination of the content of oligonucleotide sequences. The data acquisition relies on the natural process of base pairing. It is possible to determine the content of complementary oligosequences in the target DNA by the process of hybridization with oligonucleotide probes of known sequences.

  18. Identification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization.

    Science.gov (United States)

    Selvarajah, Shamini; Yoshimoto, Maisa; Maire, Georges; Paderova, Jana; Bayani, Jane; Squire, Jeremy A; Zielenska, Maria

    2007-11-01

    Osteosarcoma (OS) is an aggressive bone tumor characterized by complex abnormal karyotypes and a high level of genomic instability. Using high-resolution array comparative genomic hybridization (aCGH), a novel class of localized copy number variations called microaberrations has been detected. These genomic anomalies typically involve DNA imbalances affecting 700 kb to 1 Mb DNA, and are often associated with some type of genetic syndromes. Because the origin of instability in OS is poorly understood, we used aCGH to determine whether microaberrations were a characteristic of four OS cell lines: U-2 OS, HOS, MG-63, and SAOS-2. TP53 is mutated in SAOS-2, a line in which 17 microaberrations were found. In contrast, U-2 OS, which has a wild-type TP53, had only six such anomalies, the lowest incidence. A 500-kb microaberration within a region of gain at 5p15.33 in SAOS-2 was confirmed by fluorescence in situ hybridization. Significantly, this genomic location is close to the TERT gene, a region of gain in all four cell lines. To our knowledge, this is the first systematic analysis of the incidence of microaberrations in OS. The high levels of these anomalies detected suggest that the instability processes in OS that lead to a highly abnormal karyotypes may also be associated with acquisition of genomic microaberrations.

  19. 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

    for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation...

  20. A novel fluorescent probe: europium complex hybridized T7 phage.

    Science.gov (United States)

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  1. 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

  2. Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells ...... mismatches may be detected in the PCR and typing for HLA-DP gene variants, which differ for only one base, may be performed.......The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells...... was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must...

  3. Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

    Institute of Scientific and Technical Information of China (English)

    Paul W Anderson; Bud C Tennant; Zhenghong Lee

    2006-01-01

    AIM: To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography (PET) imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma.METHODS: Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips(R). Ten genes were selected for validation using quantitative RT-PCR and literature review was made.RESULTS: Testis enhanced gene transcript (BAX Inhibitor 1), alpha-fetoprotein, isocitrate dehydrogenase 3 (NAD+) beta, acetyl-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most downregulated genes.CONCLUSION: Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

  4. Large Scale Explorative Oligonucleotide Probe Selection for Thousands of Genetic Groups on a Computing Grid: Application to Phylogenetic Probe Design Using a Curated Small Subunit Ribosomal RNA Gene Database

    Directory of Open Access Journals (Sweden)

    Faouzi Jaziri

    2014-01-01

    Full Text Available Phylogenetic Oligonucleotide Arrays (POAs were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI. We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.

  5. Steady-State Fluorescence and Lifetime Emission Study of pH-Sensitive Probes Based on i-motif Forming Oligonucleotides Single and Double Labeled with Pyrene

    Directory of Open Access Journals (Sweden)

    Anna Dembska

    2015-09-01

    Full Text Available Cytosine-rich nucleic acids undergo pH-stimulated structural transitions leading to formation of an i-motif architecture at an acidic pH. Thus, i-motifs are good foundation for designing simple pH-sensitive fluorescent probes. We report here steady-state and time-resolved fluorescence studies of pyrene-labeled probes based on RET sequence: C4GC4GC4GC4TA (RET21, AC4GC4GC4GC4TA (RET21A and C4GC4GC4GC4T (RET20. Comparative studies with single- and double-labeled i-motif probes were carried out. For each probe, we have measured fluorescence spectra and decays for emission wavelength of 390 nm over a wide range of pH (from 4.0 to 8.0. Effect of the oligonucleotide sequence and the number of pyrene labels on the spectral characteristics of probes were discussed.

  6. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

    Science.gov (United States)

    Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

    2016-11-01

    Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

  7. Highly sensitive electrochemical sensor for mercury(II) ions by using a mercury-specific oligonucleotide probe and gold nanoparticle-based amplification.

    Science.gov (United States)

    Zhu, Zhiqiang; Su, Yuanyuan; Li, Jiang; Li, Di; Zhang, Jiong; Song, Shiping; Zhao, Yun; Li, Genxi; Fan, Chunhai

    2009-09-15

    We report a highly sensitive electrochemical sensor for the detection of Hg(2+) ions in aqueous solution by using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe and gold nanoparticles (Au NPs)-based signal amplification. The MSO probe contains seven thymine bases at both ends and a "mute" spacer in the middle, which, in the presence of Hg(2+), forms a hairpin structure via the Hg(2+)-mediated coordination of T-Hg(2+)-T base pairs. The thiolated MSO probe is immobilized on Au electrodes to capture free Hg(2+) in aqueous media, and the MSO-bound Hg(2+) can be electrochemically reduced to Hg(+), which provides a readout signal for quantitative detection of Hg(2+). This direct immobilization strategy leads to a detection limit of 1 microM. In order to improve the sensitivity, MSO probe-modified Au NPs are employed to amplify the electrochemical signals. Au NPs are comodified with the MSO probe and a linking probe that is complementary to a capture DNA probe immobilized on gold electrodes. We demonstrated that this Au NPs-based sensing strategy brings about an amplification factor of more than 3 orders of magnitude, leading to a limit of detection of 0.5 nM (100 ppt), which satisfactorily meets the sensitivity requirement of U.S. Environmental Protection Agency (EPA). This Au NPs-based Hg(2+) sensor also exhibits excellent selectivity over a spectrum of interference metal ions. Considering the high sensitivity and selectivity of this sensor, as well as the cost-effective and portable features of electrochemical techniques, we expect this Au NPs amplified electrochemical sensor will be a promising candidate for field detection of environmentally toxic mercury.

  8. Highly Sensitive Fluorometric Hg2+ Biosensor with a Mercury(Ⅱ)-Specific Oligonucleotide (MSO) Probe and Water-Soluble Graphene Oxide (WSGO)

    Institute of Scientific and Technical Information of China (English)

    Liu Xingfen; Miao Likun; Jiang Xu; Ma Yanwen; Fan Quli; Huang Wei

    2011-01-01

    A highly sensitive and selective, "turn-on" and simple Hg2+ biosensor is reported by using water-soluble gra-phene oxide (WSGO) and dye-labeled mercury(Ⅱ)-specific oligonucleotide (MSO) probe. The probe is rich of thymine (T) and can readily form the stem-loop structure which consists of the T-Hg2+-T configuration. In the ab-sence of Hg2+, the probe exists as a random coil conformation which can be readily adsorbed on the surface of WSGO by strong noncovalent binding of bases, as a result, the fluorescence of the dye labeled on the terminus of the MSO is strongly quenched by the efficient electron/energy transfer from the dye to WSGO. Upon addition of Hg2+, the formation of the T-Hg2+-T structure releases the MSO from the surface of WSGO, resulting in a restora-tion of the fluorescence of dye-labeled MSO probe. Based on this observation, a highly sensitive and selective Hg2+ sensor is developed, which can work with "turn-on" mode in aqueous solutions at room temperature. By using the fluorometric method, the limit of detection for Hg2+ can reach picomolar range (187 pmol·L-1), and it is demonstrated that the biosensor is highly selective and only minimally perturbed by a wide range of non-specific metal ions.

  9. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Science.gov (United States)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  10. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Institute of Scientific and Technical Information of China (English)

    TANG Xianghai; YU Rencheng; ZHOU Mingjiang; YU Zhigang

    2012-01-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs).This species consists of many strains that differ in their ability to produce toxins but have similar morphology,making identification difficult.In this study,species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A.minutum from two phylogenetic clades.We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes.Three ribotype-specific probes,M-GC-1,M-PC-2,and M-PC-3,were designed.The former is specific for the GC clade (“Global clade”) of A.minutum,the majority of which have been found non-toxic,and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade (“Pacific clade”).The specificity of these three probes was confirmed by FISH.All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions.However,the accessibility of rRNA molecules in ribosomes varied among the probe binding positions.Thus,there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1,M-PC-3),or just nucleolus (M-PC-2).Our results provide a methodological basis for studying the biogeography and population dynamics of A.minutum,and providing an early warning of toxic HABs.

  11. OligoHeatMap (OHM): an online tool to estimate and display hybridizations of oligonucleotides onto DNA sequences.

    Science.gov (United States)

    Croce, Olivier; Chevenet, François; Christen, Richard

    2008-07-01

    The efficiency of molecular methods involving DNA/DNA hybridizations depends on the accurate prediction of the melting temperature (T(m)) of the duplex. Many softwares are available for T(m) calculations, but difficulties arise when one wishes to check if a given oligomer (PCR primer or probe) hybridizes well or not on more than a single sequence. Moreover, the presence of mismatches within the duplex is not sufficient to estimate specificity as it does not always significantly decrease the T(m). OHM (OligoHeatMap) is an online tool able to provide estimates of T(m) for a set of oligomers and a set of aligned sequences, not only as text files of complete results but also in a graphical way: T(m) values are translated into colors and displayed as a heat map image, either stand alone or to be used by softwares such as TreeDyn to be included in a phylogenetic tree. OHM is freely available at http://bioinfo.unice.fr/ohm/, with links to the full source code and online help.

  12. Transcript-based redefinition of grouped oligonucleotide probe sets using AceView: High-resolution annotation for microarrays

    Directory of Open Access Journals (Sweden)

    Cam Margaret C

    2007-03-01

    Full Text Available Abstract Background Extracting biological information from high-density Affymetrix arrays is a multi-step process that begins with the accurate annotation of microarray probes. Shortfalls in the original Affymetrix probe annotation have been described; however, few studies have provided rigorous solutions for routine data analysis. Results Using AceView, a comprehensive human transcript database, we have reannotated the probes by matching them to RNA transcripts instead of genes. Based on this transcript-level annotation, a new probe set definition was created in which every probe in a probe set maps to a common set of AceView gene transcripts. In addition, using artificial data sets we identified that a minimal probe set size of 4 is necessary for reliable statistical summarization. We further demonstrate that applying the new probe set definition can detect specific transcript variants contributing to differential expression and it also improves cross-platform concordance. Conclusion We conclude that our transcript-level reannotation and redefinition of probe sets complement the original Affymetrix design. Redefinitions introduce probe sets whose sizes may not support reliable statistical summarization; therefore, we advocate using our transcript-level mapping redefinition in a secondary analysis step rather than as a replacement. Knowing which specific transcripts are differentially expressed is important to properly design probe/primer pairs for validation purposes. For convenience, we have created custom chip-description-files (CDFs and annotation files for our new probe set definitions that are compatible with Bioconductor, Affymetrix Expression Console or third party software.

  13. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

    Science.gov (United States)

    Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

  14. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

    Directory of Open Access Journals (Sweden)

    Yong Xue

    Full Text Available Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

  15. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    Science.gov (United States)

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  16. Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina

    2013-01-01

    the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages...

  17. 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs.

    Science.gov (United States)

    Hurek, T; Burggraf, S; Woese, C R; Reinhold-Hurek, B

    1993-11-01

    Phylogenetic analyses after reverse transcriptase sequencing of 16S rRNA of nitrogen-fixing, grass-associated Azoarcus strains confirmed their affiliation to the beta subdivision of the Proteobacteria. Strains representing three different species formed a phylogenetically coherent unit related to Rhodocyclus purpureus, with actual percent similarities among the three sequences ranging from 93.1 to 97.3%. Within variable regions V2 and V5, we found stretches of sequences considerably conserved within the genus Azoarcus but differing from most other gram-negative bacteria, with the specificity being enhanced when different regions were combined. Genus-specific primers selected from both regions amplified fragments from all but one Azoarcus species in polymerase chain reactions (PCR) but not from any reference strain tested. Primers of lesser specificity generated fragments from members of all five Azoarcus species as well as from some reference strains. Those unspecific amplifications could be differentiated by oligonucleotide hybridization, detecting only fragments generated from Azoarcus strains except strain 6a3, which represents the same group which could not be detected by genus-specific PCR. Thus we propose the application of PCR amplification with 16S rRNA-targeted, genus-specific primers in combination with hybridization of a 16S rRNA-targeted oligonucleotide to PCR-generated fragments as diagnostic tests; this allows an initial screening for presence of members of the genus Azoarcus.

  18. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

    2006-01-01

    A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

  19. Application of locked nucleic acid-based probes in fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Carvalho, Daniel R; Guimarães, Nuno

    2016-01-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe) RNA modifications have...... on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type...

  20. Design and application of oligonucleotide probes for fluorescent in situ identification of the filamentous bacterial morphotype Nostocoida limicola in activated sludge.

    Science.gov (United States)

    Liu, J R; Seviour, R J

    2001-09-01

    16S rRNA targeted probes, designed using sequence data from pure cultures of the three morphotypes of the filamentous bulking bacteria Nostocoida limicola I, II and III and their successful application to the in situ identification of these bacteria in activated sludge biomass samples are described here. Two probes were required to detect all the sequenced N. limicola II isolates. Results from fluorescent in situ hybridization suggest that the morphotypes N. limicola I and II contain at least two phylogenetically unrelated bacteria. The N. limicola II filaments that did not respond to the probes designed in this study fluoresced instead with the probes previously designed for the alpha-Proteobacteria. The data also suggest that both N. limicola I and III can exist in activated sludge as single, paired or clumped cells and thus in a form not recognizable microscopically as this morphotype. Some N. limicola II filaments which responded to the probes designed here were much thinner than the filaments conventionally 'identified' as this morphotype and better fitted the descriptions often used in the literature for N. limicola I.

  1. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  2. 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...

  3. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleicacid probes and tyramide signal amplification

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli N.; Nolting, Dorrit; Andersen, Lars Dyrskjøt;

    2007-01-01

    The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of mi......RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously...... protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution....

  4. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleicacid probes and tyramide signal amplification

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli N.; Nolting, Dorrit; Andersen, Lars Dyrskjøt

    2007-01-01

    RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously...... been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH...... protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution....

  5. A novel route for immobilization of oligonucleotides onto modified silica nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Rao, Kota Sreenivasa [Institute of Scientific and Industrial Research, Sanken, Osaka University, Osaka (Japan)]. E-mail: kotas_1999@yahoo.com; Rani, Sikhakolli Usha [Institute of Scientific and Industrial Research, Sanken, Osaka University, Osaka (Japan); Charyulu, Devarayapalli Kamakshaiah [Institute of Advanced Energy, Kyoto University, Gokasho, Uji (Japan); Kumar, Kamisetty Nagendra [Institute of Advanced Energy, Kyoto University, Gokasho, Uji (Japan); Lee, Bong-Kuk [Institute of Scientific and Industrial Research, Sanken, Osaka University, Osaka (Japan); Lee, Hea-Yeon [Institute of Scientific and Industrial Research, Sanken, Osaka University, Osaka (Japan); Kawai, Tomoji [Institute of Scientific and Industrial Research, Sanken, Osaka University, Osaka (Japan)

    2006-08-25

    A novel approach for immobilization of probe oligonucleotides that uses zirconium phosphate modified silica nanoparticles is proposed. The surface modification of nanoparticles was carried out in two stages. Initially binding of Zr{sup 4+} to the surface of silica nanoparticles and later treated with phosphoric acid for terminal phosphate groups. Oligonucleotide probes modified with amine group at 5'-end were strongly binds to the phosphate terminated silica nanoparticles with imidazole in presence of 0.1 mol L{sup -1} EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide], as phosphate groups are more reactive towards amine group. Various studies, i.e., synthesis of silica nanoparticles, their surface modification, probe immobilization, measurement of hybridization and effect of bovine serum albumin (BSA) were carried out during optimization of reaction conditions. The significant reduction in the background signal was observed by treating the probe modified silica nanoparticles with bovine serum albumin prior to hybridization. The probe modified silica nanoparticles were retained their properties and the hybridization was induced by exposure of single-stranded DNA (ssDNA) containing silica nanoparticles to the complementary DNA in solution. The decrease in the fluorescence signal for one mismatch and three mismatch was observed upon hybridization of probe with target DNAs, while there was no response for the random target ssDNA under the same experimental conditions. The intensity of fluorescence signal was linear to the concentration of target DNA ranging from 3.9 x 10{sup -9} to 3.0 x 10{sup -6} mol L{sup -1}. A detection limit of 1.22 x 10{sup -9} mol L{sup -1} of oligonucleotides can be estimated. The proposed hybridization assay is simple and possesses good analytical characteristics and it can provide an effective and efficient route in the development of DNA biosensors and biochips.

  6. nuID: a universal naming scheme of oligonucleotides for Illumina, Affymetrix, and other microarrays

    Directory of Open Access Journals (Sweden)

    Kibbe Warren A

    2007-05-01

    Full Text Available Abstract Background Oligonucleotide probes that are sequence identical may have different identifiers between manufacturers and even between different versions of the same company's microarray; and sometimes the same identifier is reused and represents a completely different oligonucleotide, resulting in ambiguity and potentially mis-identification of the genes hybridizing to that probe. Results We have devised a unique, non-degenerate encoding scheme that can be used as a universal representation to identify an oligonucleotide across manufacturers. We have named the encoded representation 'nuID', for nucleotide universal identifier. Inspired by the fact that the raw sequence of the oligonucleotide is the true definition of identity for a probe, the encoding algorithm uniquely and non-degenerately transforms the sequence itself into a compact identifier (a lossless compression. In addition, we added a redundancy check (checksum to validate the integrity of the identifier. These two steps, encoding plus checksum, result in an nuID, which is a unique, non-degenerate, permanent, robust and efficient representation of the probe sequence. For commercial applications that require the sequence identity to be confidential, we have an encryption schema for nuID. We demonstrate the utility of nuIDs for the annotation of Illumina microarrays, and we believe it has universal applicability as a source-independent naming convention for oligomers. Reviewers This article was reviewed by Itai Yanai, Rong Chen (nominated by Mark Gerstein, and Gregory Schuler (nominated by David Lipman.

  7. The detection of HBV DNA with gold nanoparticle gene probes

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

    Objective:Gold nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Methods:Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number of thiol-derivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solution was determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides immobilized on a nylon membrane surface acting as capturing probes,HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results:Compared with bare Au nanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environment and the absorbance peak of modified Au nanoparticles shifted from 520nm to 524nm. For Au nanoparticles, the maximal oligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was (132 ± 10) oligonucleotides per nanoparticle, and the percentage of hybridization strands on nanoparticles was (22 ± 3% ). Based on a two-probe sandwich hybridization/nanoparticle amplification/silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10-11 mol/L composite HBV DNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules in liquid. Conclusion:Our results showed that successfully prepared Au nanoparticle HBV DNA gene probes could be used to

  8. Probing hybrid modified gravity by stellar motion around Galactic Centre

    CERN Document Server

    Borka, D; Jovanović, P; Jovanović, V Borka

    2015-01-01

    We consider possible signatures for the so called {\\it hybrid gravity} within the Galactic Central Parsec. This modified theory of gravity consists of a superposition of the metric Einstein-Hilbert Lagrangian with an $f(R)$ term constructed {\\it \\`{a} la Palatini } and can be easily reduced to an equivalent scalar-tensor theory. The present analysis is based on the S2 star orbital precession around the massive compact dark object at the Galactic Centre where the simulated orbits in hybrid modified gravity are compared with astronomical observations. These simulations result with strong constraints on the range of hybrid gravity interaction parameter $\\phi_0$ and show that its most probable value, in the case of S2 star, is around -0.0009 to -0.0002. At the same time, we are also able to obtain reliable constrains on the effective mass parameter $m_{\\phi}$ of hybrid modified gravity. Its most probable value, in the case of S2 star, is around -0.0034 to -0.0025. Furthermore, the hybrid gravity potential induces...

  9. Predicting oligonucleotide-directed mutagenesis failures in protein engineering.

    Science.gov (United States)

    Wassman, Christopher D; Tam, Phillip Y; Lathrop, Richard H; Weiss, Gregory A

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.

  10. Brightness through Local Constraint-LNA-Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking

    DEFF Research Database (Denmark)

    Hövelmann, Felix; Gaspar, Imre; Loibl, Simon

    2014-01-01

    Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT......) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic...

  11. Fiber-based hybrid probe for non-invasive cerebral monitoring in neonatology

    Science.gov (United States)

    Rehberger, Matthias; Giovannella, Martina; Pagliazzi, Marco; Weigel, Udo; Durduran, Turgut; Contini, Davide; Spinelli, Lorenzo; Pifferi, Antonio; Torricelli, Alessandro; Schmitt, Robert

    2015-07-01

    Improved cerebral monitoring systems are needed to prevent preterm infants from long-term cognitive and motor restrictions. Combining advanced near-infrared diffuse spectroscopy measurement technologies, time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) will introduce novel indicators of cerebral oxygen metabolism and blood flow for neonatology. For non-invasive sensing a fiber-optical probe is used to send and receive light from the infant head. In this study we introduce a new fiber-based hybrid probe that is designed for volume production. The probe supports TRS and DCS measurements in a cross geometry, thus both technologies gain information on the same region inside the tissue. The probe is highly miniaturized to perform cerebral measurements on heads of extreme preterm infants down to head diameters of 6cm. Considerations concerning probe production focus on a reproducible accuracy in shape and precise optical alignment. In this way deviations in measurement data within a series of probes should be minimized. In addition to that, requirements for clinical use like robustness and hygiene are considered. An additional soft-touching sleeve made of FDA compatible silicone allows for a flexible attachment with respect to the individual anatomy of each patient. We present the technical concept of the hybrid probe and corresponding manufacturing methods. A prototype of the probe is shown and tested on tissue phantoms as well as in vivo to verify its operational reliability.

  12. Phenylethynylpyrene excimer forming hybridization probes for fluorescence SNP detection

    DEFF Research Database (Denmark)

    Prokhorenko, Igor A.; Astakhova, Irina V.; Momynaliev, Kuvat T.

    2009-01-01

    Excimer formation is a unique feature of some fluorescent dyes (e.g., pyrene) which can be used for probing the proximity of biomolecules. Pyrene excimer fluorescence has previously been used for homogeneous detection of single nucleotide polymorphism (SNP) on DNA. 1-Phenylethynylpyrene (1-1-PEPy...

  13. Non-hybridization saturable mechanisms play a role in the uptake of (68)Ga-Labeled LNA-DNA mixmer antisense oligonucleotides in rats.

    Science.gov (United States)

    Lendvai, Gabor; Monazzam, Azita; Velikyan, Irina; Eriksson, Barbro; Josephsson, Raymond; Långström, Bengt; Bergström, Mats; Estrada, Sergio

    2009-09-01

    Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specific for rat chromogranin-A mRNA was labeled with (68)Ga and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribonucleotides. In addition, uptake studies of (68)Ga-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of (68)Ga-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.

  14. In situ hybridization with labeled probes: assessment of african Swine Fever virus in formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Ballester, Maria; Rodríguez, Fernando

    2015-01-01

    In situ hybridization (ISH) has become a very valuable molecular diagnostic tool to detect specific DNA or RNA sequences in biological samples through the use of complementary DNA- or RNA-labeled probes. Here, we describe an optimized in situ hybridization protocol to detect African swine fever virus (ASFV) DNA in formalin-fixed, paraffin-embedded tissues using digoxigenin-labeled probes.

  15. Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

    Science.gov (United States)

    Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

    2017-03-01

    This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

  16. Hybridization-based biosensor containing hairpin probes and use thereof

    Science.gov (United States)

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  17. Edge turbulence measurement in Heliotron J using a combination of hybrid probe system and fast cameras

    Energy Technology Data Exchange (ETDEWEB)

    Nishino, N., E-mail: nishino@hiroshima-u.ac.jp [Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima (Japan); Zang, L. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Takeuchi, M. [JAEA, Naka, Ibaraki (Japan); Mizuuchi, T.; Ohshima, S. [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Kasajima, K.; Sha, M. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Mukai, K. [NIFS, Toki, Gifu (Japan); Lee, H.Y. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Nagasaki, K.; Okada, H.; Minami, T.; Kobayashi, S.; Yamamoto, S.; Konoshima, S.; Nakamura, Y.; Sano, F. [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan)

    2013-07-15

    The hybrid probe system (a combination of Langmuir probes and magnetic probes), fast camera and gas puffing system were installed at the same toroidal section to study edge plasma turbulence/fluctuation in Heliotron J, especially blob (intermittent filament). Fast camera views the location of the probe head, so that the probe system yields the time evolution of the turbulence/fluctuation while the camera images the spatial profile. Gas puff at the same toroidal section was used to control the plasma density and simultaneous gas puff imaging technique. Using this combined system the filamentary structure associated with magnetic fluctuation was found in Heliotron J at the first time. The other kind of fluctuation was also observed at another experiment. This combination measurement enables us to distinguish MHD activity and electro-static activity.

  18. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

  19. Probing Structure and Composition of Nickel/Titanium Carbide Hybrid Interfaces at the Atomic Scale (Preprint)

    Science.gov (United States)

    2010-01-01

    The transition in structure and composition across the titanium carbide /nickel hybrid interface has been determined at near atomic resolution by...coupling high-resolution transmission electron microscopy with three-dimensional atom probe tomography. The titanium carbide phase adopts a rocksalt-type

  20. Problems connected with the use of oligonucleotide probes with a high degree of degeneracy. Identification of mRNA and of cDNA clones corresponding to the gene of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.E.; Grishin, A.V.; Arsenyan, S.G.; Broude, N.E.; Grinkevich, V.A.; Filippova, L.Yu.; Severtsova, I.V.; Modyanov, N.N.

    1986-10-01

    To identify and search for nucleotide sequences containing the structural part of the gene of the ..cap alpha..-subunit of Na/sup +/, K/sup +/-ATPase, 17-membered oligonucleotide probes corresponding to the peptide Lys-Asp-Ala-Phe-Gln-Asn have been synthesized. It has been shown that, with a 64-fold degeneracyd, the 17-membered probe is suitable only for the identification of a specific sequence in mRNA. To search for clones containing cDNA fragments, preliminary fractionation of the probes with the aid of HPLC or the resynthesis of groups of oligonucleotides with a lower degeneracy is necessary.

  1. Combining gene expression data from different generations of oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Kong Sek

    2004-10-01

    Full Text Available Abstract Background One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. Results We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. Conclusions Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different

  2. 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.

  3. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    OpenAIRE

    de los Reyes, F L; Ritter, W; Raskin, L.

    1997-01-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacteri...

  4. Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

    Directory of Open Access Journals (Sweden)

    Lanfranchi Gerolamo

    2002-10-01

    Full Text Available Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences. This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene, with each probe evaluated for hybridization kinetics (Tm and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41% were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39% could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

  5. Chromosomal Aberrations in ETV6/RUNX1-positive Childhood Acute Lymphoblastic Leukemia using 244K Oligonucleotide Array Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Zakaria Zubaidah

    2012-11-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is a heterogeneous form of hematological cancer consisting of various subtypes. We are interested to study the genetic aberration in precursor B-cell ALL with specific t(12;21 translocation in childhood ALL patients. A high resolution 244K array-based Comparative Genomic Hybridization (array-CGH was used to study eleven ETV6/RUNX1-positive childhood acute lymphoblastic leukemia (ALL patients. Result 155 chromosomal aberrations (119 losses, 36 gains were reported in the array findings, corresponding to 76.8% deletions and 23.2% amplifications. The ETV6 gene deletion occurred in 4 of the patients, corresponding to 45% of the sample. The most common alterations above 1 Mb were deletion 6q (13%, 12p (12% and 9p (8%, and duplication 4q (6% and Xq (4%. Other genes important in ALL were also identified in this study including RUNX1, CDKN2A, FHIT, and PAX5. The array-CGH technique was able to detect microdeletion as small as 400 bp. Conclusion The results demonstrate the usefulness of high resolution array-CGH as a complementary tool in the investigation of ALL.

  6. A comparison of hybridization efficiency between flat glass and channel glass solid supports.

    Science.gov (United States)

    Betanzos-Cabrera, Gabriel; Harker, Brent W; Doktycz, Mitchel J; Weber, James L; Beattie, Kenneth L

    2008-01-01

    Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

  7. Ultrathin oligonucleotide layers for fluorescence-based DNA sensors

    Science.gov (United States)

    Furch, M.; Ueberfeld, J.; Hartmann, Andreas; Bock, Daniel; Seeger, Stefan

    1996-11-01

    Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.

  8. A 16S rRNA-targeted Probe for Detection of Lactobacilli and Enterococci in Faecal Samples by Fluorescent In Situ Hybridization

    OpenAIRE

    Harmsen, Hermie J. M.; Elfferich, Peter; Schut, Frits; Welling, Gjalt W

    2011-01-01

    A group-specific 16S rRNA-targeted oligonucleotide probe S-G-Lab-0158-a-A20 (Lab158) was designed and validated to quantify species of the phylogenetic group lactobacilli-enterococci. The Lab158 probe detects nearly all species of the genera Lactobacillus, Enterococcus, Pediococcus, Weissella, Vagococcus, Leuconostoc and Oenococcus. The specificity of the probe was tested on various species of the target group and on a range of common intestinal bacteria. For these experiments, procedures to ...

  9. Delivery of antisense oligonucleotides using cholesterol-modified sense dendrimers and cationic lipids

    NARCIS (Netherlands)

    Chaltin, Patrick; Margineanu, Anca; Marchand, Damien; Aerschot, Arthur Van; Rozenski, Jef; Schryver, Frans De; Herrmann, Andreas; Müllen, Klaus; Juliano, Rudolph; Fisher, Michael H.; Kang, Hyunmin; Feyter, Steven De; Herdewijn, Piet

    2005-01-01

    Cholesterol modified mono-, di-, and tetrameric oligonucleotides were synthesized and hybridized with antisense oligonucleotides to study their incorporation in cationic liposomes together with the influence of this dendrimeric delivery system on biological activity. Electrostatic interactions seem

  10. Identification of cDNAs by direct hybridization using cosmid probes

    Energy Technology Data Exchange (ETDEWEB)

    Lennon, G.G.; Lieuallen, K.

    1993-12-01

    The goal of this effort is to quickly obtain as many chromosome-specific cDNAs with as much map and sequence detail as possible. Many techniques have been proposed to isolate and identify genes within defined genomic regions; the technique discussed here is direct hybridization of a relatively complex genomic probe, an entire cosmid clone, to cDNA libraries. This method continues to be a straightforward technique with a fair number of successes.

  11. Probe hybridization array typing: a binary typing method for Escherichia coli.

    Science.gov (United States)

    Srinivasan, U; Zhang, L; France, A M; Ghosh, D; Shalaby, W; Xie, J; Marrs, C F; Foxman, B

    2007-01-01

    The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., "library on a slide"), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

  12. Detection and Analysis of DNA Hybridization Characteristics by using Thermodynamic Method

    Energy Technology Data Exchange (ETDEWEB)

    Kim, D.K.; Kwon, Y.S. [Donga University, Pusan (Korea)

    2002-06-01

    The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and application area. So, the improvement of DNA hybridization detection method is very important for the determination of this hybridization reaction. Several molecular biological techniques require accurate predictions of matched versus mismatched hybridization thermodynamics, such as PCR, sequencing by hybridization, gene diagnostics and antisense oligonucleotide probes. In addition, recent developments of oligonucleotide chip arrays as means for biochemical assays and DNA sequencing requires accurate knowledge of hybridization thermodynamics and population ratios at matched and mismatched target sites. In this study, we report the characteristics of the probe and matched, mismatched target oligonucleotide hybridization reaction using thermodynamic method. Thermodynamic of 5 oligonucleotides with central and terminal mismatch sequences were obtained by measured UV-absorbance as a function of temperature. The data show that the nearest-neighbor base-pair model is adequate for predicting thermodynamics of oligonucleotides with average deviations for {delta}H{sup O}, {delta}S{sup O}, {delta}G{sub 37}{sup O} and T{sub m}, respectively. (author). 6 refs., 4 figs., 5 tabs.

  13. Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

    Institute of Scientific and Technical Information of China (English)

    Xiang-Rong Tang; Ji-Shen Zhang; Hui Zhao; Yu-Hua Gong; Yong-Zhong Wang; Jian-Long Zhao

    2007-01-01

    AIM: To determine the genotype distribution of hepatitis B virus (HBV) with a newly oligonucleotide chip assay among the HBV carriers in Eastern China.METHODS: An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing.RESULTS: HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for8.3% (n = 33), 83.2% (n = 333), and 8.5% (n = 34),respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing.CONCLUSION: The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.

  14. 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.

  15. Uprobe 2008: an online resource for universal overgo hybridization-based probe retrieval and design.

    Science.gov (United States)

    Sullivan, Robert T; Morehouse, Caroline B; Thomas, James W

    2008-07-01

    Cross-species sequence comparisons are a prominent method for analyzing genomic DNA and an ever increasing number of species are being selected for whole-genome sequencing. Targeted comparative genomic sequencing is a complementary approach to whole-genome shotgun sequencing and can produce high-quality sequence assemblies of orthologous chromosomal regions of interest from multiple species. Genomic libraries necessary to support targeted mapping and sequencing projects are available for more than 90 vertebrates. An essential step for utilizing these and other genomic libraries for targeted mapping and sequencing is the development of the hybridization-based probes, which are necessary to screen a genomic library of interest. The Uprobe website (http://uprobe.genetics.emory.edu) provides a public online resource for identifying or designing 'universal' overgo-hybridization probes from conserved sequences that can be used to efficiently screen one or more genomic libraries from a designated group of species. Currently, Uprobe provides the ability to search or design probes for use in broad groups of species, including mammals and reptiles, as well as more specific clades, including marsupials, carnivores, rodents and nonhuman primates. In addition, Uprobe has the capability to design custom probes from multiple-species sequence alignments provided by the user, thus providing a general tool for targeted comparative physical mapping.

  16. Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

    Science.gov (United States)

    Souza, Elaine; Nascimento, Gustavo; Santana, Nataly; Ferreira, Danielly; Lima, Manoel; Natividade, Edna; Martins, Danyelly; Lima-Filho, José

    2011-01-01

    A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. PMID:22163916

  17. Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

    Science.gov (United States)

    McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

    2015-07-01

    The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

  18. A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

    Institute of Scientific and Technical Information of China (English)

    YAN QIN LU; JIN XIANG HAN; ZHONG LIN SHEN; CHUAN XI WANG

    2006-01-01

    A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker,while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction,probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the muin these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.

  19. [Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

    Science.gov (United States)

    Bogomolov, A G; Karamysheva, T V; Rubtsov, N B

    2014-01-01

    A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

  20. Chaotic mixer improves microarray hybridization.

    Science.gov (United States)

    McQuain, Mark K; Seale, Kevin; Peek, Joel; Fisher, Timothy S; Levy, Shawn; Stremler, Mark A; Haselton, Frederick R

    2004-02-15

    Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.

  1. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    Directory of Open Access Journals (Sweden)

    John S. Furey

    2005-04-01

    Full Text Available In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA and Hierarchical Cluster Analysis (HCA were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  2. Direct microcontact printing of oligonucleotides for biochip applications

    Directory of Open Access Journals (Sweden)

    Trévisiol E

    2005-07-01

    Full Text Available Abstract Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting

  3. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  4. Carrier-phonon interactions in hybrid halide perovskites probed with ultrafast anisotropy studies

    Science.gov (United States)

    Rivett, Jasmine P. H.; Richter, Johannes M.; Price, Michael B.; Credgington, Dan; Deschler, Felix

    2016-09-01

    Hybrid halide perovskites are at the frontier of optoelectronic research due to their excellent semiconductor properties and solution processability. For this reason, much attention has recently been focused on understanding photoexcited charge-carrier generation and recombination in these materials. Conversely, very few studies have so far been devoted to understanding carrier-carrier and carrier-phonon scattering mechanisms in these materials. This is surprising given that carrier scattering mechanisms fundamentally limit charge-carrier motilities and therefore the performance of photovoltaic devices. We apply linear polarization selective transient absorption measurements to polycrystalline CH3NH3PbBr3 hybrid halide perovskite films as an effective way of studying the scattering processes in these materials. Comparison of the photo induced bleach signals obtained when the linear polarizations of the pump and probe are aligned either parallel or perpendicular to one another, reveal a significant difference in spectral intensity and shape within the first few hundred femtoseconds after photoexcitation.

  5. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe.

    Science.gov (United States)

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. [Stain hybridization method with pRepHind probe for the diagnosis of Plasmodium falciparum].

    Science.gov (United States)

    Moleón Borodowsky, I

    1992-01-01

    A study was conducted on the parasitemia detection level and the specificity of the pRepHind DNA probe for diagnosing Plasmodium falciparum by the stain hybridization method. The parasitemia detection level was studied by using dilutions of a P. falciparum in vitro culture, adjusted by direct microscopic examination to 1; 0.1; 0.01; 0.001; 0.0001 and 0.00001% of parasited red cells. Specificity was increased by using DNA extractions from P. Yoelii, P. berghei and human leucocytes. The results showed that the method was able to detect 0.0001% of parasitemia starting from DNA extractions of 100 L infected red cells. The pRepHind probe only detected specifically DNA from P. falciparum. It is concluded that the method is suitable for being used in the diagnosis of infection due to P. falciparum.

  7. Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography

    DEFF Research Database (Denmark)

    Lolas, Ihab Bishara Lolas; Chen, Xijuan; Bester, Kai

    2012-01-01

    Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA......-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions...... of enrichment culture incubated with 13C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting...

  8. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification.

    Science.gov (United States)

    Silahtaroglu, Asli N; Nolting, Dorrit; Dyrskjøt, Lars; Berezikov, Eugene; Møller, Morten; Tommerup, Niels; Kauppinen, Sakari

    2007-01-01

    The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

  9. Preliminary test of an imaging probe for nuclear medicine using hybrid pixel detectors

    CERN Document Server

    Bertolucci, Ennio; Mettivier, G; Montesi, M C; Russo, P

    2002-01-01

    We are investigating the feasibility of an intraoperative imaging probe for lymphoscintigraphy with Tc-99m tracer, for sentinel node radioguided surgery, using the Medipix series of hybrid detectors coupled to a collimator. These detectors are pixelated semiconductor detectors bump-bonded to the Medipix1 photon counting read-out chip (64x64 pixel, 170 mu m pitch) or to the Medipix2 chip (256x256 pixel, 55 mu m pitch), developed by the European Medipix collaboration. The pixel detector we plan to use in the final version of the probe is a semi-insulating GaAs detector or a 1-2 mm thick CdZnTe detector. For the preliminary tests presented here, we used 300-mu m thick silicon detectors, hybridized via bump-bonding to the Medipix1 chip. We used a tungsten parallel-hole collimator (7 mm thick, matrix array of 64x64 100 mu m circular holes with 170 mu m pitch), and a 22, 60 and 122 keV point-like (1 mm diameter) radioactive sources, placed at various distances from the detector. These tests were conducted in order ...

  10. Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant RNA Viruses and a Viroid

    Institute of Scientific and Technical Information of China (English)

    Zhiyou DU; Bo JIN; Wenhong LIU; Liang CHEN; Jishuang CHEN

    2007-01-01

    In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing 32P labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA.The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a 32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore,potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.

  11. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems.

    Science.gov (United States)

    Varizhuk, Anna M; Kaluzhny, Dmitry N; Novikov, Roman A; Chizhov, Alexandr O; Smirnov, Igor P; Chuvilin, Andrey N; Tatarinova, Olga N; Fisunov, Gleb Y; Pozmogova, Galina E; Florentiev, Vladimir L

    2013-06-21

    New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.

  12. Optical response of a quantum dot-metal nanoparticle hybrid interacting with a weak probe field.

    Science.gov (United States)

    Kosionis, Spyridon G; Terzis, Andreas F; Sadeghi, Seyed M; Paspalakis, Emmanuel

    2013-01-30

    We study optical effects in a hybrid system composed of a semiconductor quantum dot and a spherical metal nanoparticle that interacts with a weak probe electromagnetic field. We use modified nonlinear density matrix equations for the description of the optical properties of the system and obtain a closed-form expression for the linear susceptibilities of the quantum dot, the metal nanoparticle, and the total system. We then investigate the dependence of the susceptibility on the interparticle distance as well as on the material parameters of the hybrid system. We find that the susceptibility of the quantum dot exhibits optical transparency for specific frequencies. In addition, we show that there is a range of frequencies of the applied field for which the susceptibility of the semiconductor quantum dot leads to gain. This suggests that in such a hybrid system quantum coherence can reverse the course of energy transfer, allowing flow of energy from the metallic nanoparticle to the quantum dot. We also explore the susceptibility of the metal nanoparticle and show that it is strongly influenced by the presence of the quantum dot.

  13. A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

    Directory of Open Access Journals (Sweden)

    Klein Harvey

    2003-08-01

    Full Text Available Abstract Detection of unknown single nucleotide polymorphism (SNP relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos, the other includes oligos specific for known SNP within the same genomic region (variant-oligos. Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.

  14. Detection of Glucose with Atomic Absorption Spectroscopy by Using Oligonucleotide Functionalized Gold Nanoparticle.

    Science.gov (United States)

    Zhang, Hong; Yan, Honglian; Ling, Liansheng

    2016-06-01

    A novel method for the detection of glucose was established with atomic absorption spectroscopy by using the label of gold nanoparticle (AuNP). Silver-coated glass assembled with oligonucleotide 5'-SH-T12-AGA CAA GAG AGG-3' (Oligo 1) was acted as separation probe, oligonucleotide 5'-CAA CAG AGA ACG-T12-SH-3' modified gold nanoparticle (AuNP-Oligo 2) was acted as signal-reporting probe. Oligonucleotide 5'-CGT TCT CTG TTG CCT CTC TTG TCT-3' (Oligo 3) could hybridize with Oligo 1 on the surface of silver-coated glass and AuNP-Oligo 2, and free AuNP-Oligo 2 could be removed by rinsing with buffer. Hence the concentration of Oligo 3 was transformed into the concentration of gold element. In addition, Oligo 3 could be cleaved into DNA fragments by glucose, glucose oxidase and Fe(2+)-EDTA through Fenton reaction. Thereby the concentration of glucose could be transformed to the absorbance of gold element. Under the optimum conditions, the integrated absorbance decreased proportionally to the concentration of glucose over the range from 50.0 μM to 1.0 mM with a detection limit of 40.0 μM. Moreover, satisfactory result was obtained when the assay was used to determinate glucose in human serum.

  15. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    . By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r...

  16. De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

    Science.gov (United States)

    Maydan, Jason S.; Okada, H. Mark; Flibotte, Stephane; Edgley, Mark L.; Moerman, Donald G.

    2009-01-01

    Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome. PMID:19189945

  17. A universal oligonucleotide microarray with a minimal number of probes for the detection and identification of viroids at the genus level.

    Directory of Open Access Journals (Sweden)

    Yongjiang Zhang

    Full Text Available A major challenge in the agricultural industry is the development of techniques that can screen plant samples for viroid infection. Microarrays are promising in this regard, as their high throughput nature can potentially allow for the detection of a range of viroids in a single test. In this paper we present a microarray that can detect a wide spectrum of all 8 reported viroid genera including 37 known plant viroid species. The array was constructed using an automated probe design protocol which generated a minimal number of probes to detect viroids at the genus level. The designed microarray showed a high specificity and sensitivity when tested with a set of standard virus samples. Finally, the microarray was applied to screen infected field samples, with Hop stunt viroid infection identified as the major disease causing pathogen for an infected citrus sample.

  18. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno

    2015-01-01

    acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization......In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo...

  19. Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

    Science.gov (United States)

    Stender, H; Kurtzman, C; Hyldig-Nielsen, J J; Sørensen, D; Broomer, A; Oliveira, K; Perry-O'Keefe, H; Sage, A; Young, B; Coull, J

    2001-02-01

    A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

  20. Submolecular Structure and Orientation of Oligonucleotide Duplexes Tethered to Gold Electrodes Probed by Infrared Reflection Absorption Spectroscopy: Effect of the Electrode Potentials.

    Science.gov (United States)

    Kékedy-Nagy, László; Ferapontova, Elena E; Brand, Izabella

    2017-02-23

    at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.

  1. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    Science.gov (United States)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  2. A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2'-O-Methyl RNA + LNA

    DEFF Research Database (Denmark)

    Søe, Martin Jensen; Møller, Trine; Dufva, Martin;

    2011-01-01

    The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show...... that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared...... to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number mi...

  3. A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

    DEFF Research Database (Denmark)

    Perelle, S.; Josefsen, Mathilde Hartmann; Hoorfar, Jeffrey

    2004-01-01

    Cycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving...

  4. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    Science.gov (United States)

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

  5. Gold Nanoparticle-based Layer-by-Layer Enhancement of DNA Hybridization Electrochemical Signal at Carbon Nanotube Modified Carbon Paste Electrode

    Institute of Scientific and Technical Information of China (English)

    Li Bo NIE; Jian Rong CHEN; Yu Qing MIAO; Nong Yue HE

    2006-01-01

    Colloid gold nanoparticle-based layer-by-layer amplification approach was applied to enhance the electrochemical detection sensitivity of DNA hybridization at carbon nanotube modified carbon paste electrodes (CNTPEs). Streptavidin was immobilized onto the surface of CNTPEs, and the conjugation of biotin labeled target oligonucleotides to the above immobilized streptavidin was performed, followed by the hybridization of target oligonucleotides with the gold nanoparticle-labeled DNA probe and then the layer-by-layer enhanced connection of gold nanoparticles, on which oligonucleotides complementary to the DNA probe were attached, to the hybridization system. The differential pulse voltammetry (DPV) signal of total gold nanoparticles was monitored. It was found that the layer-by-layer colloidal gold DPV detection enhanced the sensitivity by about one order of magnitude compared with that of one-layer detection. One-base mismatched DNA and complementary DNA could be distinguished clearly.

  6. Spatial Distribution of Escherichia coli in the Mouse Large Intestine Inferred from rRNA In Situ Hybridization

    DEFF Research Database (Denmark)

    Poulsen, Lars Kærgaard; Lan, Fusheng; Sternberg, Claus

    1994-01-01

    Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from th...

  7. Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

    Institute of Scientific and Technical Information of China (English)

    MENG Juan; GU Qin-sheng; LIN Shi-ming; PENG Bin; LIU Li-feng; TIAN Yan-ping; LI Li

    2007-01-01

    Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops,Zuccini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Papaya ringspot virus watermelon strain(PRSV-W)and Squash mosaic virus(SqMV),as a good alternative assay in seed health test and epidemiological and transgenic research.Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves.And three SqMV probes of different lengths(0.55,1.6,and 2.7 kb,respectively)were designed to investigate the effect of hybridization.The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W,and SqMV was down to 1:160,1:160,1:320,1:160,and 1:320,respectively.Three SqMV probes of different length showed no differences on the sensitivity and specificity.The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities,sensitivities,specificity,and reproducibilities.

  8. Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

    NARCIS (Netherlands)

    Rosado, A.S.; Seldin, L.; Wolters, A.; Elsas, van J.D.

    1996-01-01

    A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific o

  9. Probing nanoscale chemical segregation and surface properties of antifouling hybrid xerogel films.

    Science.gov (United States)

    Destino, Joel F; Gatley, Caitlyn M; Craft, Andrew K; Detty, Michael R; Bright, Frank V

    2015-03-24

    Over the past decade there has been significant development in hybrid polymer coatings exhibiting tunable surface morphology, surface charge, and chemical segregation-all believed to be key properties in antifouling (AF) coating performance. While a large body of research exists on these materials, there have yet to be studies on all the aforementioned properties in a colocalized manner with nanoscale spatial resolution. Here, we report colocalized atomic force microscopy, scanning Kelvin probe microscopy, and confocal Raman microscopy on a model AF xerogel film composed of 1:9:9 (mol:mol:mol) 3-aminopropyltriethoxysilane (APTES), n-octyltriethoxysilane (C8), and tetraethoxysilane (TEOS) formed on Al2O3. This AF film is found to consist of three regions that are chemically and physically unique in 2D and 3D across multiple length scales: (i) a 1.5 μm thick base layer derived from all three precursors; (ii) 2-4 μm diameter mesa-like features that are enriched in free amine (from APTES), depleted in the other species and that extend 150-400 nm above the base layer; and (iii) 1-2 μm diameter subsurface inclusions within the base layer that are enriched in hydrogen-bonded amine (from APTES) and depleted in the other species.

  10. Evaluating a hybrid three-dimensional metrology system: merging data from optical and touch probe devices

    Science.gov (United States)

    Gerde, Janice R.; Christens-Barry, William A.

    2011-08-01

    In a project to meet requirements for CBP Laboratory analysis of footwear under the Harmonized Tariff Schedule of the United States (HTSUS), a hybrid metrology system comprising both optical and touch probe devices has been assembled. A unique requirement must be met: To identify the interface-typically obscured in samples of concern-of the "external surface area upper" (ESAU) and the sole without physically destroying the sample. The sample outer surface is determined by discrete point cloud coordinates obtained using laser scanner optical measurements. Measurements from the optically inaccessible insole region are obtained using a coordinate measuring machine (CMM). That surface similarly is defined by point cloud data. Mathematically, the individual CMM and scanner data sets are transformed into a single, common reference frame. Custom software then fits a polynomial surface to the insole data and extends it to intersect the mesh fitted to the outer surface point cloud. This line of intersection defines the required ESAU boundary, thus permitting further fractional area calculations to determine the percentage of materials present. With a draft method in place, and first-level method validation underway, we examine the transformation of the two dissimilar data sets into the single, common reference frame. We also will consider the six previously-identified potential error factors versus the method process. This paper reports our on-going work and discusses our findings to date.

  11. 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

  12. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

    Science.gov (United States)

    Peccia, J; Marchand, E A; Silverstein, J; Hernandez, M

    2000-07-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

  13. Using an aryl phenanthroimidazole moiety as a conjugated flexible intercalator to improve the hybridization efficiency of a triplex-forming oligonucleotide.

    Science.gov (United States)

    Osman, Amany M A; Jørgensen, Per T; Bomholt, Niels; Pedersen, Erik B

    2008-12-01

    When inserting 2-phenyl or 2-naphth-1-yl-phenanthroimidazole intercalators (X and Y, respectively) as bulges into triplex-forming oligonucleotides, both intercalators show extraordinary high thermal stability of the corresponding Hoogsteen-type triplexes and Hoogsteen-type parallel duplexes with high discrimination to Hoogsteen mismatches. Molecular modeling shows that the phenyl or the naphthyl ring stacks with the nucleobases in the TFO, while the phenanthroimidazol moiety stacks with the base pairs of the dsDNA. DNA-strands containing the intercalator X show higher thermal triplex stability than DNA-strands containing the intercalator Y. The difference can be explained by a lower degree of planarity of the intercalator in the case of naphthyl. It was also observed that triplex stability was considerably reduced when the intercalators X or Y was replaced by 2-(naphthlen-1-yl)imidazole. This confirms intercalation as the important factor for triplex stabilization and it rules out an alternative complexation of protonated imidazole with two phosphate groups. The intercalating nucleic acid monomers X and Y were obtained via a condensation reaction of 9,10-phenanthrenequinone (4) with (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde (3a) or (S)-4-(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)-1-naphthaldehyde (3b), respectively, in the presence of acetic acid and ammonium acetate. The required monomers for DNA synthesis using amidite chemistry were obtained by standard deprotection of the hydroxy groups followed by 4,4'-dimethoxytritylation and phosphitylation.

  14. Minimum probe length for unique identification of all open reading frames in a microbial genome

    Energy Technology Data Exchange (ETDEWEB)

    Sokhansanj, B A; Ng, J; Fitch, J P

    2000-03-05

    In this paper, we determine the minimum hybridization probe length to uniquely identify at least 95% of the open reading frame (ORF) in an organism. We analyze the whole genome sequences of 17 species, 11 bacteria, 4 archaea, and 2 eukaryotes. We also present a mathematical model for minimum probe length based on assuming that all ORFs are random, of constant length, and contain an equal distribution of bases. The model accurately predicts the minimum probe length for all species, but it incorrectly predicts that all ORFs may be uniquely identified. However, a probe length of just 9 bases is adequate to identify over 95% of the ORFs for all 15 prokaryotic species we studied. Using a minimum probe length, while accepting that some ORFs may not be identified and that data will be lost due to hybridization error, may result in significant savings in microarray and oligonucleotide probe design.

  15. The delivery of therapeutic oligonucleotides.

    Science.gov (United States)

    Juliano, Rudolph L

    2016-08-19

    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. © The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. 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.

  17. Antisense oligonucleotides in cancer.

    Science.gov (United States)

    Castanotto, Daniela; Stein, Cy A

    2014-11-01

    Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood.

  18. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    DEFF Research Database (Denmark)

    1997-01-01

    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide wit......, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and $i(in vitro) mutagenesis....

  19. Experimental characterization of the lower hybrid wave field on the first pass using a magnetic probe array

    Science.gov (United States)

    Shinya, T.; Baek, S. G.; Wallace, G. M.; Parker, R. R.; Shiraiwa, S.; Takase, Y.

    2016-10-01

    Experimental characterization of the lower hybrid (LH) wave propagation from the launcher to the core plasma is important to validate an antenna spectrum model and to identify parasitic wave-edge plasma interactions occurring in front of the launcher. On Alcator C-Mod, the wave frequency spectrum and dominant parallel wavenumber are characterized with two probe arrays installed near the edge plasma. The first one is mounted on a radially movable structure that is about 108 deg toroidally away from the launcher. A phasing scan experiment at moderate density suggests a resonance-cone propagation of the launched slow LH wave with a finite spectral width. As plasma density is raised, the measured power decreases, correlated with the observed loss of efficiency. Recently, the second probe array with an increased number of probes has been installed on a limiter that is 54 deg. toroidally away from the launcher, which is expected to be dominantly sensitive to the wave-field directly leaving the launcher. An initial measurement shows that the probe array detects a coherent wave field. A full-wave model to evaluate the wave electric-field pattern in front of the probe array is under development. If available, further experimental and modeling results will be presented. Supported by USDoE Award(s) DE-FC02-99ER54512 and Japan/U.S. Cooperation in Fusion Research and Development.

  20. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

    Directory of Open Access Journals (Sweden)

    Shiu Lin-Yi

    2011-04-01

    Full Text Available Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. Results A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26 and 97.7% (43/44, respectively. Conclusions Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

  1. Design and application of 60mer oligonucleotide microarray in SARS coronavirus detection

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute respiratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submitted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as restricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microarray for hybridization. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microarray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide microarray, which can improve the positive ratio of the diagnosis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.

  2. A comparative analysis of existing oligonucleotides selection ...

    African Journals Online (AJOL)

    SERVER

    2007-07-04

    Jul 4, 2007 ... In system biology, DNA microarray technology is an indispensable tool for the ... the microarray probes are of critical importance for the hybridization experiments as ... at unraveling molecular systems (the function of the gen-.

  3. Towards Fluorescence In Vivo Hybridization (FIVH Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

    Directory of Open Access Journals (Sweden)

    Sílvia Fontenete

    Full Text Available In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA/ 2' O-methyl RNA (2'OMe probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH. In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.

  4. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    Science.gov (United States)

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  5. LINE-1 repetitive DNA probes for species-specific cloning from Mus spretus and Mus domesticus genomes.

    Science.gov (United States)

    Rikke, B A; Hardies, S C

    1991-12-01

    Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.

  6. Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Zhang Zheng

    2007-12-01

    Full Text Available Abstract Background The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens. Results The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity. In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/μL and 103 cfu/mL per reaction. Conclusion The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

  7. Specific detection of very low concentrations of DNA oligonucleotides with DNA-coated long-period grating biosensor

    Science.gov (United States)

    Czarnecka, Karolina H.; Dominik, Magdalena; Janczuk-Richter, Marta; Niedziółka-Jönsson, Joanna; Bock, Wojtek J.; Śmietana, Mateusz

    2017-04-01

    Label-free biosensing using optical long-period gratings (LPGs) induced in optical fiber and coated with biological compound enables for detection and monitoring the kinetics of reactions taking places on the sensor's surface. The labelfree detection effect for the LPG biosensor working near the dispersion turning point (DTP) of higher order cladding modes can be observed as resonance wavelength shift induced by binding of biomolecules to fiber surface. In this study we analyzed shift of the resonance wavelength for LPG after its functionalization with DNA layer and during DNA hybridization. It has seen found that the LPG is able to be functionalized with DNA oligonucleotide, acting as a probe and after the functionalization can selectively bind specific complementary DNA oligonucleotides in a very low concentration. DNA-coated LPG can be used as a probe capturing specific DNA sequences with wide ranges of concentrations from 0.1 to 10 pM. Hybridization of the DNA on the fiber surface has also been verified with Midorii Green florescent marker.

  8. Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis

    Directory of Open Access Journals (Sweden)

    Lipkin Alexey

    2007-05-01

    Full Text Available Abstract Background the use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then. Results here we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of Agelena orientalis species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from Agelena orientalis, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same Agelena material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of Agelena orientalis spider contains a much larger family of related toxin-like sequences than previously thought. Conclusion with upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.

  9. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    Directory of Open Access Journals (Sweden)

    Michael Liew

    2016-01-01

    Full Text Available Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe and MYC 8;14 translocation using IGH-MYC (a fusion probe. Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas.

  10. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    Science.gov (United States)

    Liew, Michael; Rowe, Leslie; Clement, Parker W.; Miles, Rodney R.; Salama, Mohamed E.

    2016-01-01

    Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH) is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe) and MYC 8;14 translocation using IGH-MYC (a fusion probe). Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas. PMID:27217970

  11. Specific detection of Lawsonia intracellularis in porcine proliferative enteropathy inferred from fluorescent rRNA in situ hybridization

    DEFF Research Database (Denmark)

    Boye, Mette; Jensen, Tim Kåre; Møller, Kristian;

    1998-01-01

    of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L. intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy. We used 10 tissue samples from pigs without proliferative mucosal changes as negative...... controls. The results showed that the oligonucleotide probe is specific for L. intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L. intracellularis in formalin-fixed tissue.......Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy. A specific oligonucleotide probe was designed and the specificity...

  12. Construction of a repeat-free dual color fluorescent in situ hybridization probe for ROS1 gene in non-small cell lung cancer diagnosis

    Institute of Scientific and Technical Information of China (English)

    程弘夏

    2014-01-01

    Objective To establish a repeat-free ROS1 gene fluorescence in situ hybridization(FISH)probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequene.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared

  13. Lipid Oligonucleotide Conjugates as Responsive Material

    Science.gov (United States)

    2012-09-28

    U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS Amphiphiles, oligonucleotides, lipids...peer-reviewed journals: (c) Presentations 1. Philippe Barthélémy, « Hybrid Lipids for Biomedical Applications », Targeting and Triggering Basic Research ...Steadel C. ; Pierre, N. ; Barthélémy, P. : Oligonucléotides amphiphile : Journée Scientifique de l’IFR 66, Talence, le 2 décembre 2008, France 29. Taib

  14. Formation of a hybrid plasmonic waveguide mode probed by dispersion measurement

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H. [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Quantum Nanoelectronics Research Center, Tokyo Institute of Technology, Oh-Okayama, Meguro-ku, Tokyo 152-8551 (Japan); Kurata, H., E-mail: kurata@eels.kuicr.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2015-04-07

    Hybrid waveguides, i.e., dielectric waveguides combined with plasmonic waveguides, have great potential for concomitantly exhibiting subwavelength confinement and long range propagation, enabling a highly integrated photonic circuit. We report the characterization of hybrid waveguide modes excited in Si/SiO{sub 2}/Al films, by dispersion measurement using angle-resolved electron energy-loss spectroscopy. This experiment directly verifies the formation of the hybrid waveguide mode with a strongly localized electromagnetic field in a 6-nm-thick SiO{sub 2} layer. The results clearly describe the characteristic behavior of the hybrid waveguide mode, which depends on the effective index of the constituent dielectric waveguide and the surface plasmon-polariton modes.

  15. Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid Agent Development

    Directory of Open Access Journals (Sweden)

    Uwe Seibold

    2014-01-01

    Full Text Available Molecular imaging—and especially positron emission tomography (PET—has gained increasing importance for diagnosis of various diseases and thus experiences an increasing dissemination. Therefore, there is also a growing demand for highly affine PET tracers specifically accumulating and visualizing target structures in the human body. Beyond the development of agents suitable for PET alone, recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI, as this combination of modalities can provide clinical advantages. PET, due to the high tissue penetration of the γ-radiation emitted by PET nuclides, allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body. OI on the contrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumor margins and infected lymph nodes during surgery without bearing a radiation burden for the surgeon. Thus, there is an emerging interest in bimodal agents for PET and OI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseases. This short review summarizes the available hybrid probes developed for dual PET and OI and discusses future directions for hybrid agent development.

  16. Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays

    Directory of Open Access Journals (Sweden)

    Khan Shehnaz

    2004-02-01

    Full Text Available Abstract Background Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. Results Significantly (p M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p Conclusions This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

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

    Science.gov (United States)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Ahmad, Haslina; Heng, Lee Yook; Karim, Nurul Huda Abd; Harun, Siti Norain

    2014-09-01

    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)2(PIP)]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)2(PIP)]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)2(PIP)]2+ with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  18. The Preparation of Magnetic Silica Nanospheres and Incorporation of CdSe/ZnS Quantum Dots-DNA Probe.

    Science.gov (United States)

    Do, Youngjin; Kim, Jongsung

    2016-03-01

    Silica nanospheres containing magnetic particles were prepared, and CdSe/ZnS QDs functionalized with carboxyl group were incorporated into the silica nanospheres by EDC/NHS coupling reaction. The silica nanospheres were prepared by a co-precipitation of ferrous and ferric solutions followed by the sol-gel reaction of TEOS (tetraethoxysilane) and APTES (3-aminopropyltriethoxysilane) using base catalyst. The size of magnetic silica nanospheres was confirmed by Transmission electron microscope (TEM). Thiol group modified single stranded oligonucleotides were immobilized on the surface of QDs and fluorescence quenching by intercalation dye (TOTO-3) after hybridization with target oligonucleotide was observed. The fluorescence from QDs could be quenched by intercalating dye (TOTO-3) after hybridization of target DNA to probe DNA. This shows that the magnetic silica-QD-DNA probe can be used to detect specific DNA.

  19. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  20. Fusobacterium necrophorum determined as abortifacient in sheep by laser capture microdissection and fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Boye, Mette; Aalbæk, Bent; Agerholm, Jørgen S.

    2006-01-01

    at late pregnancy by a technique that combines laser capture microdissection (LCM) and fluorescent in situ hybridization (LCM-FISH). Cultural bacteriological examination had failed to identify an infectious agent but by histological examination, large colonies of bacteria associated with tissue......Fluorescent in situ hybridization (FISH) has been extensively used for identification of individual microbial cells within their natural environment. The present work describes the identification of Fusobacterium necrophorum in formalin-fixed tissue samples from three sets of ovine twins aborted......RNA-targeting oligonucleotide probe specific for F. necrophorum was used in a FISH assay. In situ hybridization showed a high density of F. necrophorum in all examined tissue sections. Simultaneous probing with a general bacterial probe EUB338 and the specific probe for F. necrophorum showed that no other bacteria could...

  1. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for iNOS mRNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Erik F.J. de E-mail: e.f.j.de.vries@pet.azg.nl; Vroegh, Joke; Dijkstra, Gerard; Moshage, Han; Elsinga, Philip H.; Jansen, Peter L.M.; Vaalburg, Willem

    2004-07-01

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[{sup 18}F]fluorobenzyl)-2-bromoacetamide. Cellular uptake and efflux of oligonucleotide complexed with FuGENE-6 were rapid, unlike naked oligonucleotide, which hardly accumulated. However, neither uptake nor efflux showed any selectivity for iNOS expressing cells. The oligonucleotide showed a high level of non-specific binding, which may have obscured its specific hybridization to iNOS mRNA.

  2. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  3. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    Science.gov (United States)

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions.

  4. 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.

  5. Computationally Probing the Performance of Hybrid, Heterogeneous, and Homogeneous Iridium-Based Catalysts for Water Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    García-Melchor, Max [SUNCAT Center for Interface Science and Catalysis, Department of Chemical Engineering, Stanford University, Stanford CA (United States); Vilella, Laia [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST),Tarragona (Spain); Departament de Quimica, Universitat Autonoma de Barcelona, Barcelona (Spain); López, Núria [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST), Tarragona (Spain); Vojvodic, Aleksandra [SUNCAT Center for Interface Science and Catalysis, SLAC National Accelerator Laboratory, Menlo Park CA (United States)

    2016-04-29

    An attractive strategy to improve the performance of water oxidation catalysts would be to anchor a homogeneous molecular catalyst on a heterogeneous solid surface to create a hybrid catalyst. The idea of this combined system is to take advantage of the individual properties of each of the two catalyst components. We use Density Functional Theory to determine the stability and activity of a model hybrid water oxidation catalyst consisting of a dimeric Ir complex attached on the IrO2(110) surface through two oxygen atoms. We find that homogeneous catalysts can be bound to its matrix oxide without losing significant activity. Hence, designing hybrid systems that benefit from both the high tunability of activity of homogeneous catalysts and the stability of heterogeneous systems seems feasible.

  6. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  7. In situ hybridization of cytokine mRNA using alkaline phosphatase-labelled oligodeoxynucleotide probes

    DEFF Research Database (Denmark)

    Clausen, Bettina Hjelm; Fenger, Christina; Finsen, B.

    2013-01-01

    In situ hybridization is a powerful tool for visualizing cellular gene expression in morphologically preserved brain tissue giving precise information on the regional expression of specific mRNA sequences in cells of diverse phenotype. Here, we describe a sensitive, simple, and robust method using...

  8. Hybrid intracerebral probe with integrated bare LED chips for optogenetic studies.

    Science.gov (United States)

    Ayub, Suleman; Gentet, Luc J; Fiáth, Richárd; Schwaerzle, Michael; Borel, Mélodie; David, François; Barthó, Péter; Ulbert, István; Paul, Oliver; Ruther, Patrick

    2017-09-01

    This article reports on the development, i.e., the design, fabrication, and validation of an implantable optical neural probes designed for in vivo experiments relying on optogenetics. The probes comprise an array of ten bare light-emitting diode (LED) chips emitting at a wavelength of 460 nm and integrated along a flexible polyimide-based substrate stiffened using a micromachined ladder-like silicon structure. The resulting mechanical stiffness of the slender, 250-μm-wide, 65-μm-thick, and 5- and 8-mm-long probe shank facilitates its implantation into neural tissue. The LEDs are encapsulated by a fluropolymer coating protecting the implant against the physiological conditions in the brain. The electrical interface to the external control unit is provided by 10-μm-thick, highly flexible polyimide cables making the probes suitable for both acute and chronic in vivo experiments. Optical and electrical properties of the probes are reported, as well as their in vivo validation in acute optogenetic studies in transgenic mice. The depth-dependent optical stimulation of both excitatory and inhibitory neurons is demonstrated by altering the brain activity in the cortex and the thalamus. Local network responses elicited by 20-ms-long light pulses of different optical power (20 μW and 1 mW), as well as local modulation of single unit neuronal activity to 1-s-long light pulses with low optical intensity (17 μW) are presented. The ability to modulate neural activity makes these devices suitable for a broad variety of optogenetic experiments.

  9. Using NV centers to probe magnetization dynamics in normal metal/magnetic insulator hybrid system at the nanoscale

    Science.gov (United States)

    Zhang, Huiliang; Ku, Mark J. H.; Han, Minyong; Casola, Francesco; van der Sar, Toeno; Yacoby, Amir; Walsworth, Ronald L.

    2016-05-01

    Understanding magnetization dynamics induced by electric current is of great interest for both fundamental and practical reasons. Great endeavor has been dedicated to spin-orbit torques (SOT) in metallic structures, while quantitative study of analogous phenomena in magnetic insulators remains challenging where transport measurements are not feasible. Recently we have developed techniques using nitrogen vacancy (NV) centers in diamond to probe few-nanometre-scale correlated-electron magnetic excitations (i.e., spin waves). Here we demonstrate how this powerful tool can be implemented to study magnetization dynamics inside ferromagnetic insulator, Yttrium iron garnet (YIG) with spin injection from electrical current through normal metal (Platinum in our case). Particularly our work will focus on NV magnetic detection, imaging, and spectroscopy of coherent auto-oscillations in Pt/YIG microdisc. Magnetic fluctuations and local temperature measurements, both with nearby NV centers, will also be interesting topics relevant to SOT physics in Pt/YIG hybrid system.

  10. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    Science.gov (United States)

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  11. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    Science.gov (United States)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  12. Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

    NARCIS (Netherlands)

    Harmsen, HJM; Gibson, GR; Elfferich, P; Raangs, GC; Wildeboer-Veloo, ACM; Argaiz, A; Roberfroid, MB; Welling, GW

    2000-01-01

    Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared From fecal samples of three healthy volunteers. To intro

  13. QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDIZATION OF BIFIDOBACTERIUM SPP WITH GENUS-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED PROBES AND ITS APPLICATION IN FECAL SAMPLES

    NARCIS (Netherlands)

    LANGENDIJK, PS; SCHUT, F; JANSEN, GJ; RAANGS, GC; KAMPHUIS, GR; WILKINSON, MHF; WELLING, GW

    1995-01-01

    Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and VS of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide prob

  14. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  15. Probing the structure and function of biopolymer-carbon nanotube hybrids with molecular dynamics

    Science.gov (United States)

    Johnson, Robert R.

    2009-12-01

    Nanoscience deals with the characterization and manipulation of matter on the atomic/molecular size scale in order to deepen our understanding of condensed matter and develop revolutionary technology. Meeting the demands of the rapidly advancing nanotechnological frontier requires novel, multifunctional nanoscale materials. Among the most promising nanomaterials to fulfill this need are biopolymer-carbon nanotube hybrids (Bio-CNT). Bio-CNT consists of a single-walled carbon nanotube (CNT) coated with a self-assembled layer of biopolymers such as DNA or protein. Experiments have demonstrated that these nanomaterials possess a wide range of technologically useful properties with applications in nanoelectronics, medicine, homeland security, environmental safety and microbiology. However, a fundamental understanding of the self-assembly mechanics, structure and energetics of Bio-CNT is lacking. The objective of this thesis is to address this deficiency through molecular dynamics (MD) simulation, which provides an atomic-scale window into the behavior of this unique nanomaterial. MD shows that Bio-CNT composed of single-stranded DNA (ssDNA) self-assembles via the formation of high affinity contacts between DNA bases and the CNT sidewall. Calculation of the base-CNT binding free energy by thermodynamic integration reveals that these contacts result from the attractive pi--pi stacking interaction. Binding affinities follow the trend G > A > T > C. MD reveals that long ssDNA sequences are driven into a helical wrapping about CNT with a sub-10 nm pitch by electrostatic and torsional interactions in the backbone. A large-scale replica exchange molecular dynamics simulation reveals that ssDNA-CNT hybrids are disordered. At room temperature, ssDNA can reside in several low-energy conformations that contain a sequence-specific arrangement of bases detached from CNT surface. MD demonstrates that protein-CNT hybrids composed of the Coxsackie-adenovirus receptor are biologically

  16. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Michael J Lodes

    Full Text Available Bacterial and viral upper respiratory infections (URI produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

  17. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

    Science.gov (United States)

    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  18. Development of peptide nucleic acid probes for detection of the HER2 oncogene.

    Directory of Open Access Journals (Sweden)

    Belhu Metaferia

    Full Text Available Peptide nucleic acids (PNAs have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.

  19. Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications.

    Science.gov (United States)

    Beaucage, S L

    2001-08-01

    This report emphasizes the interfacial chemistry that is required to ensure proper attachment of oligonucleotides onto the surface of microarrays. For example, strategies for the covalent attachment of pre-synthesized oligonucleotides to glass slides, gold films, polyacrylamide gel pads, polypyrrole films, and optical fibers are surveyed in an attempt to better define the parameters for optimal formation and detection of DNA hybrids. These parameters include among others, the nature and length of the linkers attaching oligonucleotides to the arrays, and the surface density of oligonucleotides required for unhindered hybridization with DNA targets. Sensitive detection methods such as the use of light-scattering techniques, molecular beacons, surface plasmon resonance, attenuated total internal reflection-FTIR, and the evanescent field excitation of fluorescence from surface-bound fluorophores have been developed to study the kinetics and specificity of hybridization events. Finally, the synthesis of oligonucleotides directly on glass surfaces and polypropylene sheets has been investigated to enable DNA sequencing by hybridization and achieve oligonucleotide densities of ca. 10(6) sequences per cm(2) on DNA chips.

  20. Dry-reagent disposable biosensor for visual genotyping of single nucleotide polymorphisms by oligonucleotide ligation reaction: application to pharmacogenetic analysis.

    Science.gov (United States)

    Toubanaki, Dimitra K; Christopoulos, Theodore K; Ioannou, Penelope C; Gravanis, Achille

    2008-08-01

    Most genotyping methods for known single-nucleotide polymorphisms (SNPs) are based on hybridization with allele-specific probes, oligonucleotide ligation reaction (OLR), primer extension or invasive cleavage. OLR offers superior specificity because it involves two recognition events; namely, the hybridization of an allele-specific probe and a common probe to adjacent positions on target DNA. OLR products can be detected by microtiter well-based colorimetric, time-resolved fluorimetric or chemiluminometric assays, electrophoresis, microarrays, microspheres, and homogeneous fluorimetric or colorimetric assays. We have developed a simple, robust, and low-cost disposable biosensor in dry-reagent format, which allows visual genotyping with no need for instrumentation. The OLR mixture contains a biotinylated common probe and an allele-specific probe with a (dA)(20) segment at the 3'-end. OLR products are denatured and applied to the biosensor next to gold nanoparticles that are decorated with oligo(dT) strands. The sensor is immersed in the appropriate buffer and all components migrate by capillary action. The OLR product is captured by immobilized streptavidin at the test zone (TZ) of the sensor and hybridizes with the oligo(dT) strands of the nanoparticles. A characteristic red line is generated due to the accumulation of nanoparticles. The excess nanoparticles are captured by immobilized oligo(dA) at the control zone of the strip, giving a second red line. We have applied successfully the proposed OLR-dipstick assay to the genotyping of four SNPs in the drug-metabolizing enzyme genes CYP2D6 ((*)3 and (*)4) and CYP2C19 ((*)2 and (*)3). The results were in agreement with direct sequencing.

  1. Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

    Science.gov (United States)

    Rull, Jordi; Nonglaton, Guillaume; Costa, Guillaume; Fontelaye, Caroline; Marchi-Delapierre, Caroline; Ménage, Stéphane; Marchand, Gilles

    2015-11-01

    The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO2) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric contrast optical technique, atomic force microscopy, multiple internal reflection infrared spectroscopy and X-ray photoelectron spectroscopy. The shelf life of the 3,4-epoxybutyltrimethoxysilane coating layer has also been studied. Finally, two different strategies of NH2-oligonucleotide grafting on EBTMOS coating layer have been compared, i.e. reductive amination and nucleophilic substitution, SN2. This EBTMOS based coating layer can be used for a wide range of applications

  2. An imputation approach for oligonucleotide microarrays.

    Directory of Open Access Journals (Sweden)

    Ming Li

    Full Text Available Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.

  3. Chemically robust fluoroalkyl phthalocyanine-oligonucleotide bioconjugates and their GRP78 oncogene photocleavage activity.

    Science.gov (United States)

    Patel, Pradeepkumar; Patel, Hemantbhai H; Borland, Emily; Gorun, Sergiu M; Sabatino, David

    2014-06-18

    The first representative of functionalized fluoroalkyl phthalocyanines, F48H7(COOH)PcZn, is reported. The complex generates (1)O2 affording long-lasting photooxidation of an external substrate without self-decomposition. The carboxylic group couples with an antisense oligonucleotide targeting GRP78 oncogenes, resulting in the F48H7PcZn-cancer targeting oligonucleotide (CTO). The bioconjugated fluorophthalocyanine effectively hybridizes complementary GRP78 DNA and mRNA sequences. Piperidine cleavage assays reveal desired photochemical oligonucleotide oxidative degradation for both F48H7PcZn-CTO:DNA and F48H7PcZn-CTO:mRNA hybrids. This new materials strategy could be extended to other functional fluorinated phthalocyanines-antisense oligonucleotide combinations for long-lasting oncogene-targeting photodynamic therapy.

  4. Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

    Science.gov (United States)

    Tønjum, T; Caugant, D A; Bøvre, K

    1992-12-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.

  5. Fast hybridization solution for the detection of immobilized nucleic acids.

    Science.gov (United States)

    Yang, T T; Kain, S R

    1995-03-01

    We have developed a fast hybridization solution, termed ExpressHyb, for the rapid and sensitive detection of nucleic acids immobilized on membrane supports. This solution reduces typical hybridization times of 12-24 h to as little as 1 h while simultaneously increasing the sensitivity of detection in many applications. Using ExpressHyb, human beta-actin mRNA was detected on a human multiple tissue Northern (MTN) blot following a 30-min hybridization, with optimal detection occurring with a 1-h hybridization interval. The moderately abundant human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was detected using similar hybridization conditions and yielded improved signal-to-background characteristics relative to overnight hybridizations in conventional solutions. ExpressHyb can be used with either 32P- or digoxigenin-labeled probes and works effectively with both cDNA and oligonucleotide probes. For non-isotopic detection in particular, ExpressHyb reduces the nonspecific background commonly encountered with this technique. In cDNA library screening, ExpressHyb was found to both reduce the time required for effective hybridizations and to increase the number of positive colonies obtained relative to conventional overnight procedures. Taken together, these results illustrate the broad capability of ExpressHyb Hybridization Solution to improve nucleic acid detection in a variety of important techniques.

  6. Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

    Science.gov (United States)

    Jarczewska, Marta; Ziółkowski, Robert; Górski, Łukasz; Malinowska, Elżbieta

    2014-04-01

    The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

  7. A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

    Directory of Open Access Journals (Sweden)

    Bidard Frédérique

    2010-06-01

    Full Text Available Abstract Background The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. Findings We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS, we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. Conclusions A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

  8. Probing particle acceleration in lower hybrid turbulence via synthetic diagnostics produced by PIC simulations

    Science.gov (United States)

    Cruz, F.; Fonseca, R. A.; Silva, L. O.; Rigby, A.; Gregori, G.; Bamford, R. A.; Bingham, R.; Koenig, M.

    2016-10-01

    Efficient particle acceleration in astrophysical shocks can only be achieved in the presence of initial high energy particles. A candidate mechanism to provide an initial seed of energetic particles is lower hybrid turbulence (LHT). This type of turbulence is commonly excited in regions where space and astrophysical plasmas interact with large obstacles. Due to the nature of LH waves, energy can be resonantly transferred from ions (travelling perpendicular to the magnetic field) to electrons (travelling parallel to it) and the consequent motion of the latter in turbulent shock electromagnetic fields is believed to be responsible for the observed x-ray fluxes from non-thermal electrons produced in astrophysical shocks. Here we present PIC simulations of plasma flows colliding with magnetized obstacles showing the formation of a bow shock and the consequent development of LHT. The plasma and obstacle parameters are chosen in order to reproduce the results obtained in a recent experiment conducted at the LULI laser facility at Ecole Polytechnique (France) to study accelerated electrons via LHT. The wave and particle spectra are studied and used to produce synthetic diagnostics that show good qualitative agreement with experimental results. Work supported by the European Research Council (Accelerates ERC-2010-AdG 267841).

  9. In situ hybridization of bat chromosomes with human (TTAGGGn probe, after previous digestion with Alu I

    Directory of Open Access Journals (Sweden)

    Karina de Cassia Faria

    2002-01-01

    Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.

  10. Anti-tumor activity of splice-switching oligonucleotides

    OpenAIRE

    Bauman, John A; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard

    2010-01-01

    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. T...

  11. Precise and selective sensing of DNA-DNA hybridization by graphene/Si-nanowires diode-type biosensors

    Science.gov (United States)

    Kim, Jungkil; Park, Shin-Young; Kim, Sung; Lee, Dae Hun; Kim, Ju Hwan; Kim, Jong Min; Kang, Hee; Han, Joong-Soo; Park, Jun Woo; Lee, Hosun; Choi, Suk-Ho

    2016-08-01

    Single-Si-nanowire (NW)-based DNA sensors have been recently developed, but their sensitivity is very limited because of high noise signals, originating from small source-drain current of the single Si NW. Here, we demonstrate that chemical-vapor-deposition-grown large-scale graphene/surface-modified vertical-Si-NW-arrays junctions can be utilized as diode-type biosensors for highly-sensitive and -selective detection of specific oligonucleotides. For this, a twenty-seven-base-long synthetic oligonucleotide, which is a fragment of human DENND2D promoter sequence, is first decorated as a probe on the surface of vertical Si-NW arrays, and then the complementary oligonucleotide is hybridized to the probe. This hybridization gives rise to a doping effect on the surface of Si NWs, resulting in the increase of the current in the biosensor. The current of the biosensor increases from 19 to 120% as the concentration of the target DNA varies from 0.1 to 500 nM. In contrast, such biosensing does not come into play by the use of the oligonucleotide with incompatible or mismatched sequences. Similar results are observed from photoluminescence microscopic images and spectra. The biosensors show very-uniform current changes with standard deviations ranging ~1 to ~10% by ten-times endurance tests. These results are very promising for their applications in accurate, selective, and stable biosensing.

  12. Localization of cannabinoid CB1 receptor mRNA using ribonucleotide probes: methods for double- and single-label in situ hybridization.

    Science.gov (United States)

    Hohmann, Andrea G

    2006-01-01

    This chapter presents a reliable, detailed method for performing double-label in situ hybridization (ISH) that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs. This method permits simultaneous detection of two different species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that are complementary to their target mRNAs. Single-label ISH may be accomplished by following the procedures described for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe. An alkaline phosphatase-dependent chromogen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Necessary controls that are required to document the specificity of the labeling of the digoxigenin and radiolabeled probes are described. The methods detailed herein may be employed to detect even low levels of a target mRNA. These methods may be utilized to study co-localization and coregulation of expression of a particular gene within identified neurons in multiple systems.

  13. Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures.

    Science.gov (United States)

    Clavé, Guillaume; Chatelain, Grégory; Filoramo, Arianna; Gasparutto, Didier; Saint-Pierre, Christine; Le Cam, Eric; Piétrement, Olivier; Guérineau, Vincent; Campidelli, Stéphane

    2014-05-01

    The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

  14. Electrochemiluminescent biosensor of ATP using tetrahedron structured DNA and a functional oligonucleotide for Ru(phen)3(2+) intercalation and target identification.

    Science.gov (United States)

    Bu, Nan-Nan; Gao, Ai; He, Xi-Wen; Yin, Xue-Bo

    2013-05-15

    Restricted target accessibility and surface-induced perturbation of the aptamer structure are the main limitations in single-stranded DNA aptamer-based electrochemical sensors. Chemical labeling of the aptamer with a probe at the end of aptamer is inefficient and time-consuming. In this work, tetrahedron-structured DNA (ts-DNA) and a functionalized oligonucleotide (FO) were used to develop an electrochemiluminescence (ECL) aptasensor with adenosine triphosphate (ATP) as a model target. The ts-DNA was formed with three thiolated oligonucleotides and one oligonucleotide containing anti-ATP aptamer. The FO contained a complementary strand to the anti-ATP aptamer and an intermolecular duplex for Ru(phen)3(2+) intercalation. After the ts-DNA was immobilized on the electrode surface through gold-thiol interactions, hybridization between the anti-ATP aptamer and its complementary strand introduced the intercalated Ru(phen)3(2+) to the electrode. ECL emission from Ru(phen)3(2+) was observed with tripropylamine as a co-reactant. Once ATP reacted with its aptamer, the aptamer-complimentary strand duplex dissociated and the intermolecular duplex containing Ru(phen)3(2+) was released. The difference in emission before and after reaction with ATP was used to quantify ATP with a detection limit of 0.2nM. The ts-DNA increased the sensitivity compared to conventional methods, and the intercalation strategy avoided a complex chemical labeling procedure.

  15. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

    Science.gov (United States)

    Kamstra, S A; Ramanna, M S; de Jeu, M J; Kuipers, A G; Jacobsen, E

    1999-01-01

    A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed.

  16. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

    Directory of Open Access Journals (Sweden)

    Magness Charles L

    2007-01-01

    Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

  18. An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

    Directory of Open Access Journals (Sweden)

    Laurenzi Ian J

    2009-12-01

    Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

  19. A statistical multiprobe model for analyzing cis and trans genes in genetical genomics experiments with short-oligonucleotide arrays

    NARCIS (Netherlands)

    Alberts, Rudi; Terpstra, Peter; Bystrykh, Leonid V.; Haan, Gerald de; Jansen, Ritsert C.

    2005-01-01

    Short-oligonucleotide arrays typically contain multiple probes per gene. In genetical genomics applications a statistical model for the individual probe signals can help in separating ‘‘true’’ differential mRNA expression from ‘‘ghost’’ effects caused by polymorphisms, misdesigned probes, and batch

  20. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    Science.gov (United States)

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera. PMID:9097423

  1. Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

    Science.gov (United States)

    Mokarram, P; Rismanchi, M; Alizadeh Naeeni, M; Mirab Samiee, S; Paryan, M; Alipour, A; Honardar, Z; Kavousipour, S; Naghibalhossaini, F; Mostafavi-Pour, Z; Monabati, A; Hosseni, S V; Shamsdin, S A

    2014-05-01

    Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

  2. 新型纳米金荧光双标survivin反义寡核苷酸探针的合成及其对人恶性黑素瘤A375细胞的影响%Synthesis of the probe of survivin antisens oligonucleotide modified gold nanoparticles and its impact on A375 cells

    Institute of Scientific and Technical Information of China (English)

    李萍; 王梅; 肖生祥; 王冰; 霍佳

    2013-01-01

    目的 利用纳米金独特的生物和光学特性,合成一种新型纳米金荧光双标survivin反义寡核苷酸探针,观察其对人恶性黑素瘤A375细胞的影响.方法 用柠檬酸钠还原法制备纳米金,将巯基修饰后的survivin反义寡核苷酸结合在纳米金表面,合成纳米金survivin反义寡核苷酸探针;再结合互补的带荧光标记的报告序列,制成纳米金荧光双标survivin反义寡核苷酸探针;将制备的探针转染人恶性黑素瘤A375细胞,荧光显微镜观察细胞内荧光图像,MTT法及流式细胞仪检测A375细胞的增殖及凋亡情况.结果 制备的纳米金荧光双标survivin反义寡核苷酸探针转染A375细胞后,可观察到清晰的细胞内荧光图像,可以抑制A375细胞的增殖,与对照组比较差异有统计学意义(P<0.05),诱导细胞凋亡增多.结论 寡核苷酸功能化的纳米金颗粒可有效转染进人恶性黑素瘤A375细胞,通过反义封闭作用诱导人恶性黑素瘤A375细胞凋亡增多.%Objective To synthesize the probe of survivin antisense oligonucleotide modified gold nanoparticles by making use of the biological and optical characteristics of gold nanoparticles and to analyze its impact on A375 cells. Methods Citrate-stabilized gold nanoparticles were prepared using published procedures. Thiol-modified oligonucleotides were added to gold colloids to make nano-probes. Then the complementary reporter sequence was combined to prepare "nano-flare" probes. A375 cells were grown with media containing nano-flares probes. Fluorescent images were observed under the fluorescence microscope. Cell proliferation was measured by MTT and cell apoptosis was detected by Annexin V/PI double staining method. Results The nano-probes could inhibit A375 cell proliferation significantly (P<0.05) and induce cell apoptosis. Conclusion The nano-probes that can be stably synthesized can inhibit proliferation of A375 cells and induce their apoptosis.

  3. Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2013-08-06

    A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

  4. A Strategy to Optimize the Oligo-Probes for Microarray-based Detection of Viruses

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    DNA microarrays have been acknowledged to represent a promising approach for the detection of viral pathogens. However, the probes designed for current arrays could cover only part of the given viral variants, that could result in false-negative or ambiguous data. If all the variants are to be covered, the requirement for more probes would render much higher spot density and thus higher cost of the arrays. Here we have developed a new strategy for oligonucleotide probe design. Using type I human immunodeficiency virus (HIV-1) tat gene as an example, we designed the array probes and validated the optimized parameters in silico. Results show that the oligo number is significantly reduced comparing with the existing methods, while specificity and hybridization efficiency remain intact. The adoption of this method in reducing the oligo numbers could increase the detection capacity for DNA microarrays, and would significantly lower the manufacturing cost for making array chips.

  5. Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

    Science.gov (United States)

    Massey, Melissa; Ancona, Mario G; Medintz, Igor L; Algar, W Russ

    2015-12-01

    Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective

  6. Molecular Mechanisms of Antisense Oligonucleotides.

    Science.gov (United States)

    Crooke, Stanley T

    2017-04-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

  7. Thermodynamics of Oligonucleotide Duplex Melting

    Science.gov (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  8. Temperature controlling system based on FISH probe hybridization facility%基于FISH探针杂交设备的温度控制系统

    Institute of Scientific and Technical Information of China (English)

    李天庆; 张化; 张婉尧

    2015-01-01

    以玻片为基础的FISH,它的变性杂交过程完全实现自动化.FISH探针杂交仪内置一个ARM7微处理器,以控制不同温度变化,满足变性、杂交和固定温度等不同模式,可预置用按键编程进去的程序(显示屏上能指导编程),方便客户调用,提高工作效率.温度控制系统是FISH探针杂交设备的核心部分,决定了杂交仪的可靠性、稳定性及精准性.%By using FISH based on slide,automation in the degeneration process of hybrid can be achieved.Installed an internal ARM7 microprocessor,the FISH probe hybridization facility can control temperature,meet different modes such as denaturation,hybridization,fixed temperature,and so on.Procedure input by means of push-button can be preset(there are guidance on display screen)to make things easy for user,as a result,work efficiency can be improved.The temperature controlling system,which is the core of FISH probe hybridization facility and ensure its reliability,stability and accuracy.

  9. Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates.

    Science.gov (United States)

    Galluzzi, Luca; Cegna, Alessandra; Casabianca, Silvia; Penna, Antonella; Saunders, Nick; Magnani, Mauro

    2011-02-01

    Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.

  10. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    DEFF Research Database (Denmark)

    Petersen, J.; Poulsen, Lena; Petronis, S.

    2008-01-01

    DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device...... is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions...... corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40 degrees C. When processed in the MTAW, probes selected without considering melting...

  11. Oligonucleotide and Long Polymeric DNA Encoding

    Energy Technology Data Exchange (ETDEWEB)

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M

    2003-11-24

    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  12. Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    Science.gov (United States)

    Wang, Xiaolong; Gou, Deming; Xu, Shuang-yong

    2010-01-01

    Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs. PMID:20062528

  13. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  14. Unlocked nucleic acids with a pyrene-modified uracil: Synthesis, hybridization studies, fluorescent properties and i-motif stability

    DEFF Research Database (Denmark)

    Perlíková, P.; Karlsen, K.K.; Pedersen, E.B.

    2014-01-01

    .2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA/DNA and DNA/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA...... intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides...... and hybridization probes....

  15. Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry.

    Science.gov (United States)

    Jafari, Safiye; Faridbod, Farnoush; Norouzi, Parviz; Dezfuli, Amin Shiralizadeh; Ajloo, Davood; Mohammadipanah, Fatemeh; Ganjali, Mohammad Reza

    2015-10-01

    A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO2NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)3](2+/3+) redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)3](2+/3+) FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10(-15) to 1 × 10(-8) mol L(-1). The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL(-1) with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)3](2+/3+) interaction with ssDNA before and after hybridization.

  16. Inhibition of HTLV-III by exogenous oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Goodchild, J.; Zamecnik, P.C.

    1989-02-21

    A method is described of detecting the presence of HTLV-III virus in a sample by demonstrating inhibition of replication of the virus in cells which are normally killed by the HTLV-III virus after the cells have been (a) combined with the sample and an oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication and capable of hybridizing with at least the highly conserved region, the highly conserved region of the HTLV-III genome being a nucleotide sequence present in the genomes of HTLV-III isolates and the oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication being complementary to a region of the HTLV-III genome.

  17. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy

    2015-05-01

    Full Text Available Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  18. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid complex as a carrier.

    Directory of Open Access Journals (Sweden)

    Yao Geng

    Full Text Available A quantum dot (QD-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS/poly(γ-glutamic acid (γ-PGA complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.

  19. Identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction: a DNA biosensor for simultaneous visual detection of both alleles.

    Science.gov (United States)

    Toubanaki, Dimitra K; Christopoulos, Theodore K; Ioannou, Penelope C; Flordellis, Christodoulos S

    2009-01-01

    Although single nucleotide polymorphisms (SNPs) can be identified by direct hybridization with allele-specific oligonucleotide probes, enzyme-based genotyping methods offer much higher specificity and robustness. Among enzymatic methods, the oligonucleotide ligation reaction (OLR) offers the highest specificity for allele discrimination because two hybridization events are required for ligation. We report the development of a DNA biosensor that offers significant advantages over currently available methods for detection of OLR products: It allows simultaneous visual discrimination of both alleles using a single ligation reaction. Detection is complete within minutes without the need for any specialized instruments. It does not involve multiple cycles of incubation and washing. The dry-reagent format minimizes the pipetting steps. The need for qualified personnel is much lower than current methods. The principle of the assay is as follows: Following PCR amplification, a single OLR is performed using a biotinylated common probe and two allele-specific probes labeled with the haptens digoxigenin and fluorescein. Ligation products corresponding to the normal and mutant allele are double-labeled with biotin and either digoxigenin or fluorescein, respectively. The products are captured by antidigoxigenin or antifluorescein antibodies, or both, that are immobilized at the two test zones of the biosensor and react with antibiotin-functionalized gold nanoparticle reporters. The excess nanoparticles bind to biotinylated albumin that is immobilized at the control zone of the biosensor. The genotype is assigned by the characteristic red lines that appear at the two test zones. The proposed DNA biosensor constitutes a significant step toward point-of-care SNP genotyping.

  20. Sex determination of bovine preimplantation embryos by oligonucleotide microarray.

    Science.gov (United States)

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin

    2013-06-01

    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  1. Discrepancy between fluorescence in situ hybridization and multiplex ligation-dependent probe amplification in orbital recurrence of uveal melanoma 26 years after enucleation.

    Science.gov (United States)

    Russo, Andrea; Rene, Cornelius; Coupland, Sarah E; Sagili, Suresh; Damato, Bertil

    2012-01-01

    Cytogenetic analysis has transformed the management of uveal melanoma in recent years and allows categorization of such tumors into low-grade tumors with a favorable prognosis and high-grade tumors that metastasize with a fatal outcome. The authors report the case of a 73-year-old man who presented with recurrent melanoma in his left socket, 26 years after enucleation for uveal melanoma. Chromosomal analysis by multiplex ligation-dependent probe amplification revealed partial loss of chromosome 3 and gains in chromosomes 6 and 8, which were missed with fluorescence in situ hybridization. The patient developed multiple liver metastases 14 months after orbital exenteration and died 8 months later. To the best of authors' knowledge, this is the first report of late recurrence of uveal melanoma after enucleation, in which multiplex ligation-dependent probe amplification chromosomal analysis has been used. The case also highlights the limitations of fluorescence in situ hybridization and the benefits of multiplex ligation-dependent probe amplification, which is more reliable at predicting survival.

  2. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for NOS mRNA expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Dijkstra, G; Moshage, H; Elsinga, PH; Jansen, PLM; Vaalburg, W

    2004-01-01

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[F-18]fluorobenzyl)-2-bromoacetamide. Cellular u

  3. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for NOS mRNA expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Dijkstra, G; Moshage, H; Elsinga, PH; Jansen, PLM; Vaalburg, W

    2004-01-01

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[F-18]fluorobenzyl)-2-bromoacetamide. Cellular u

  4. LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin.

    Science.gov (United States)

    Yan, Jing; Yuan, Ying; Mu, Runqing; Shang, Hong; Guan, Yifu

    2014-12-01

    Oligonucleotide microarray has been one of the most powerful tools in the 'Post-Genome Era' for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

  5. LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin

    Indian Academy of Sciences (India)

    Jing Yan; Ying Yuan; Runqing Mu; Hong Shang; Yifu Guan

    2014-12-01

    Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

  6. Nanoparticle probes and mid-infrared chemical imaging for DNA microarray detection.

    Science.gov (United States)

    Mossoba, Magdi M; Al-Khaldi, Sufian F; Schoen, Brianna; Yakes, Betsy Jean

    2010-11-01

    To date most mid-infrared spectroscopic studies have been limited, due to lack of sensitivity, to the structural characterization of a single oligonucleotide probe immobilized over the entire surface of a gold-coated slide or other infrared substrate. By contrast, widely used and commercially available glass slides and a microarray spotter that prints approximately 120-μm-diameter DNA spots were employed in the present work. To our knowledge, mid-infrared chemical imaging (IRCI) in the external reflection mode has been applied in the present study for the first time to the detection of nanostructure-based DNA microarrays spotted on glass slides. Alkyl amine-modified oligonucleotide probes were immobilized on glass slides that had been prefunctionalized with succinimidyl ester groups. This molecular fluorophore-free method entailed the binding of gold-nanoparticle-streptavidin conjugates to biotinylated DNA targets. Hybridization was visualized by the silver enhancement of gold nanoparticles. The adlayer of silver, selectively bound only to hybridized spots in a microarray, formed the external reflective infrared substrate that was necessary for the detection of DNA hybridization by IRCI in the present proof-of-concept study. IRCI made it possible to discriminate between diffuse and specular external reflection modes. The promising qualitative results are presented herein, and the implications for quantitative determination of DNA microarrays are discussed.

  7. Amplified fluorescent sensing of DNA using luminescent carbon dots and AuNPs/GO as a sensing platform: A novel coupling of FRET and DNA hybridization for homogeneous HIV-1 gene detection at femtomolar level.

    Science.gov (United States)

    Qaddare, Somaye Hamd; Salimi, Abdollah

    2017-03-15

    The demand for simple, sensitive, affordable, and selective DNA biosensors is willing, due to the important role of DNA detection in the areas of disease diagnostics, environment monitoring and food safety. The presented work is devoted to the fabrication of an ultrasensitive homogeneous biosensor for the detection of DNA sequences related to HIV based on fluorescence resonance energy transfer(FRET) between carbon dots(CDs) and AuNPs as nanoquenchers. CDs as fluorophore with average size 3-4nm were prepared by hydrothermal treatment of histidine. In this respect, the hybridization was occurring between the assemblies of fluorescence CDs functionalized 5-amino-labeled oligonucleotides as capture probe and label free oligonucleotides as detection probe. Due to strong fluorescence and good biocompatibility of CDs, the capture probe was covalently conjugated to CDs. In the presence of the target probe, the association between capture probe-CDs and detection probe is stronger than that between capture probe-CDs and AuNPs, leading to the release of the capture probe-CDs from AuNPs, resulting in the recovery of the fluorescence of CDs. This oligonucleotides detection probe was observed to detect target oligonucleotides specifically and sensitively in a linear range from 50.0fM to 1.0nM with a detection limit of 15fM. Furthermore, the sensitivity of this FRET strategy amplified using AuNPs/graphene oxide nanocomposite as quencher. The Sensor response indicates only the complementary sequence showing an obvious change signal in comparison to non-complementary and two bases mismatched sequences. Moreover, satisfactory results from determination of HIV DNA target in human serum were obtained showing great potential of the proposed method for real sample analysis. The proposed biosensor with highly biocompatibility and nontoxicity, can be developed for detection of other DNA biomarkers.

  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. Diseño de oligonucléotidos para el estudio de genes celulolíticos y solventogénicos en cepas colombianas de Clostridium sp. (CLOSTRIDIACEAE Oligonucleotide Probe Design for the Study of Cellulolytic and Solventogenic Genes in Colombian Clostridiumsp. strains (Clostridiaceae

    Directory of Open Access Journals (Sweden)

    Suárez Moreno Zulma Rocío

    2007-12-01

    Full Text Available El objetivo de este estudio fue analizar las rutas metabólicas para la producción de solventes y degradación de celulosa en cepas colombianas promisorias del género Clostridium. Para ello se diseñaron sondas de hibridación que sirvieran para posteriores estudios de mejoramiento genético de las cepas. Se construyó la base de datos denominada MULTICLOST en Microsoft Access® con las secuencias de 485 genes involucrados en las rutas metabólicas arriba mencionadas, provenientes de 45 especies bacterianas y 10 especies fúngicas. Los genes fueron agrupados de acuerdo al tipo de enzima y a los dominios catalíticos o de unión a sustrato en el caso de las celulasas. Cada grupo se sometió a alineamiento múltiple en ClustalW 1.83 y con base en los resultados se crearon subgrupos de similitud mayor al 50%. Se localizaron secuencias conservadas de longitud mayor a 19 nucleótidos en GeneDoc 2.6.002 y sus valores termodinámicos fueron estimados con GeneRunner v3.05, mientras que la sensibilidad y especificidad fue verificada por búsquedas en GenBank usando BLASTN 2.2.8. En total se obtuvieron 94 secuencias conservadas con las siguientes características: longitud promedio de 24 nucleótidos, Tm promedio de 65,8 ºC y contenido de (G+C entre 14,3 y 60,0%. Se determinó que ninguna de las sondas diseñadas forma estructuras secundarias estables con Tm superior a 36,1 ºC. De acuerdo a sus características y valores termodinámicos, todas las sondas podrían ser utilizadas en la construcción de un microarreglo o en reacciones de PCR para la identificación de regiones relevantes en el mejoramiento del proceso por ingeniería metabólica.The goal of the present study was to analyze the metabolic pathways involved in solvent production and cellulose consumption by promising Colombian native strains of the genus Clostridium. Therefore a set of oligonucleotide probes was designed, with the aim of analyzing potential targets for genetic

  10. DISEÑO DE OLIGONUCLEÓTIDOS PARA EL ESTUDIO DE GENES CELULOLÍTICOS Y SOLVENTOGÉNICOS EN CEPAS COLOMBIANAS DE Clostridiumsp. (CLOSTRIDIACEAE Oligonucleotide Probe Design for the Study of Cellulolytic and Solventogenic Genes in Colombian Clostridiumsp. strains (Clostridiaceae

    Directory of Open Access Journals (Sweden)

    JOSÉ DAVID MONTOYA SOLANO

    Full Text Available El objetivo de este estudio fue analizar las rutas metabólicas para la producción de solventes y degradación de celulosa en cepas colombianas promisorias del género Clostridium. Para ello se diseñaron sondas de hibridación que sirvieran para posteriores estudios de mejoramiento genético de las cepas. Se construyó la base de datos denominada MULTICLOST en Microsoft Access® con las secuencias de 485 genes involucrados en las rutas metabólicas arriba mencionadas, provenientes de 45 especies bacterianas y 10 especies fúngicas. Los genes fueron agrupados de acuerdo al tipo de enzima y a los dominios catalíticos o de unión a sustrato en el caso de las celulasas. Cada grupo se sometió a alineamiento múltiple en ClustalW 1.83 y con base en los resultados se crearon subgrupos de similitud mayor al 50%. Se localizaron secuencias conservadas de longitud mayor a 19 nucleótidos en GeneDoc 2.6.002 y sus valores termodinámicos fueron estimados con GeneRunner v3.05, mientras que la sensibilidad y especificidad fue verificada por búsquedas en GenBank usando BLASTN 2.2.8. En total se obtuvieron 94 secuencias conservadas con las siguientes características: longitud promedio de 24 nucleótidos, Tm promedio de 65,8 ºC y contenido de (G+C entre 14,3 y 60,0%. Se determinó que ninguna de las sondas diseñadas forma estructuras secundarias estables con Tm superior a 36,1 ºC. De acuerdo a sus características y valores termodinámicos, todas las sondas podrían ser utilizadas en la construcción de un microarreglo o en reacciones de PCR para la identificación de regiones relevantes en el mejoramiento del proceso por ingeniería metabólica.The goal of the present study was to analyze the metabolic pathways involved in solvent production and cellulose consumption by promising Colombian native strains of the genus Clostridium. Therefore a set of oligonucleotide probes was designed, with the aim of analyzing potential targets for genetic improvement

  11. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    Science.gov (United States)

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-02-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

  12. Observation of Lower Hybrid Current Drive Improved Confinement with a Graphite Probe at the Last Closed Flux Surface of the HT-7 Tokamak

    Institute of Scientific and Technical Information of China (English)

    徐国盛; 万宝年; 宋梅; 凌必利; 匡光力; 丁伯江

    2002-01-01

    High time resolution measurements of the electrostatic fluctuations, radial electric field Er and turbulence-induced electron flux Гe have been performed across the transition of lower hybrid current drive improved confinement with a graphite Langmuir probe array at the last closed flux surface of the HT-7 tokamak. The decrease of Гe is dominated by the suppression of fluctuation levels, which follows the change of Er. A reversal of the poloidal propagation direction of turbulence demonstrates that the poloidal propagation is dominated by Eт× Bφ drift. The enhancement of poloidal coherence accompanies the fluctuation suppression, which suggests the subtle variation of turbulence features.

  13. [Transfection of hypertrophic cardiac myocytes in vitro with (99)Tc(m)-labeled antisense miR208b oligonucleotide].

    Science.gov (United States)

    Wang, Jing; Feng, Huijuan; Ou, Yangwei; Sun, Yungang; Wu, Juqing; Chen, Pan

    2015-08-01

    To test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro. The anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined. The labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%. The (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.

  14. Split-probe hybrid femtosecond/picosecond rotational CARS for time-domain measurement of S-branch Raman linewidths within a single laser shot.

    Science.gov (United States)

    Patterson, Brian D; Gao, Yi; Seeger, Thomas; Kliewer, Christopher J

    2013-11-15

    We introduce a multiplex technique for the single-laser-shot determination of S-branch Raman linewidths with high accuracy and precision by implementing hybrid femtosecond (fs)/picosecond (ps) rotational coherent anti-Stokes Raman spectroscopy (CARS) with multiple spatially and temporally separated probe beams derived from a single laser pulse. The probe beams scatter from the rotational coherence driven by the fs pump and Stokes pulses at four different probe pulse delay times spanning 360 ps, thereby mapping collisional coherence dephasing in time for the populated rotational levels. The probe beams scatter at different folded BOXCARS angles, yielding spatially separated CARS signals which are collected simultaneously on the charge coupled device camera. The technique yields a single-shot standard deviation (1σ) of less than 3.5% in the determination of Raman linewidths and the average linewidth values obtained for N(2) are within 1% of those previously reported. The presented technique opens the possibility for correcting CARS spectra for time-varying collisional environments in operando.

  15. Interfacial transduction of nucleic acid hybridization using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Algar, W Russ; Krull, Ulrich J

    2009-01-06

    Fluorescence resonance energy transfer (FRET) using immobilized quantum dots (QDs) as energy donors was explored as a transduction method for the detection of nucleic acid hybridization at an interface. This research was motivated by the success of the QD-FRET-based transduction of nucleic acid hybridization in solution-phase assays. This new work represents a fundamental step toward the assembly of a biosensor, where immobilization of the selective chemistry on a surface is desired. After immobilizing QD-probe oligonucleotide conjugates on optical fibers, a demonstration of the retention of selectivity was achieved by the introduction of acceptor (Cy3)-labeled single-stranded target oligonucleotides. Hybridization generated the proximity required for FRET, and the resulting fluorescence spectra provided an analytical signal proportional to the amount of target. This research provides an important framework for the future development of nucleic acid biosensors based on QDs and FRET. The most important findings of this work are that (1) a QD-FRET solid-phase hybridization assay is viable and (2) a passivating layer of denatured bovine serum albumin alleviates nonspecific adsorption, ultimately resulting in (3) the potential for a reusable assay format and mismatch discrimination. In this, the first incarnation of a solid-phase QD-FRET hybridization assay, the limit of detection was found to be 5 nM, and the dynamic range was almost 2 orders of magnitude. Selective discrimination of the target was shown using a three-base-pairs mismatch from a fully complementary sequence. Despite a gradual loss of signal, reuse of the optical fibers over multiple cycles of hybridization and dehybridization was possible. Directions for further improvement of the analytical performance by optimizing the design of the QD-probe oligonucleotide interface are identified.

  16. Characterization of self-assembled DNA concatemers from synthetic oligonucleotides

    Directory of Open Access Journals (Sweden)

    Lu Sun

    2014-08-01

    Full Text Available Studies of DNA–ligand interaction on a single molecule level provide opportunities to understand individual behavior of molecules. Construction of DNA molecules with repetitive copies of the same segments of sequences linked in series could be helpful for enhancing the interaction possibility for sequence-specific binding ligand to DNA. Here we report on the use of synthetic oligonucleotides to self-assembly into duplex DNA concatemeric molecules. Two strands of synthetic oligonucleotides used here were designed with 50-mer in length and the sequences are semi-complimentary so to hybridize spontaneously into concatemers of double stranded DNA. In order to optimize the length of the concatemers the oligonucleotides were incubated at different oligomer concentrations, ionic strengths and temperatures for different durations. Increasing the salt concentration to 200 mM NaCl was found to be the major optimizing factor because at this enhanced ionic strength the concatemers formed most quickly and the other parameters had no detectable effect. The size and shape of formed DNA concatemers were studied by gel electrophoresis in agarose, polyacrylamide gels and by AFM. Our results show that linear DNA constructs up to several hundred base pairs were formed and could be separated from a substantial fraction of non-linear constructs.

  17. An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations

    DEFF Research Database (Denmark)

    Liu, Yuliang; Guðnason, Haukur; Li, Yiping

    2014-01-01

    or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable...

  18. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    is a valuable tool for a more realistic assessment of SRB abundance in the natural environment. The distribution of SRB was investigated in a coastal marine sediment by hybridization of membrane-immobilized rRNA with oligonucleotide probes. As represented by general probe-target groups, SRB rRNA contributed...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates...

  19. 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)

  20. A hydrophobic dye-encapsulated nano-hybrid as an efficient fluorescent probe for living cell imaging.

    Science.gov (United States)

    Chang, Shu; Wu, Xumeng; Li, Yongsheng; Niu, Dechao; Ma, Zhi; Zhao, Wenru; Gu, Jinlou; Dong, Wenjie; Ding, Feng; Zhu, Weihong; Shi, Jianlin

    2012-07-01

    Water-soluble hydrophobic-dye@nano-hybrids (DPN@NHs) with extraordinarily enhanced fluorescent performance were fabricated by encapsulating the hydrophobic dye molecules into the core of the hybrid nanospheres based on the self-assembly of amphiphilic block copolymers followed by shell cross-linking using 3-mercaptopropyltrimethoxy-silane. The DPN@NHs are 50 nm in size, are monodispersed in aqueous solution and have a quantum yield enhanced by 30 times.

  1. [Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip].

    Science.gov (United States)

    Griadunov, D A; Getman, I A; Chizhova, S I; Mikhaĭlovich, V M; Zasedatelev, A S; Romanov, G A

    2011-01-01

    A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.

  2. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

    Science.gov (United States)

    Kolganova, N. A.; Shchyolkina, A. K.; Chudinov, A. V.; Zasedatelev, A. S.; Florentiev, V. L.; Timofeev, E. N.

    2012-01-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  3. Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

    Energy Technology Data Exchange (ETDEWEB)

    Abel, B.; Aslan, K. [Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251 (United States)

    2012-11-15

    This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization, where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  4. Rapid Genotyping of the Human Renin (REN Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

    Directory of Open Access Journals (Sweden)

    Line Wee

    2010-01-01

    Full Text Available Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm. Ninety-two mother-father-child triads (n=276 from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs in REN. All three htSNPs (rs5705, rs1464816 and rs3795575 were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041 were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

  5. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 1; The Study for the Distance 4.5Rs

    Science.gov (United States)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2010-01-01

    Our report devotes a 3D numerical hybrid model of the interaction of the solar wind with the Solar Probe spacecraft. The Solar Probe Plus (SPP) model includes 3 main parts, namely, a non-conducting heat shield, a support system, and cylindrical section or spacecraft bus that contains the particle analysis devices and antenna. One observes an excitation of the low frequency Alfven and whistler type wave directed by the magnetic field with an amplitude of about (0.06-0.6) V/m. The compression waves and the jumps in an electric field with an amplitude of about (0.15-0.7) V/m were also observed. The wave amplitudes are comparable to or greater than previously estimated max wave amplitudes that SPP is expected to measure. The results of our hybrid simulation will be useful for understanding the plasma environment near the SPP spacecraft at the distance 4.5 Rs. Future simulation will take into account the charging of the spacecraft, the charge separation effects, an outgassing from heat shield, a photoionization and an electron impact ionization effects near the spacecraft.

  6. Specific detection of the genus Serpulina, S-hyodysenteriae and S-pilosicoli in porcine intestines by fluorescent rRNA in situ hybridization

    DEFF Research Database (Denmark)

    Boye, Mette; Jensen, Tim Kåre; Møller, Kristian;

    1998-01-01

    , for the detection of Serpulina isolates at genus level as well as for specific detection of S. hyodysenteriae and S. pilosicoli in formalin-fixed colon tissue sections from pigs suffering from swine dysentery and porcine colonic spirochaetosis, respectively. Tissue sections were also used from pigs without any......A fluorescent-labelled in situ hybridization method targeting rRNA was devised to facilitate specific identification and diagnosis of diarrhoea and colitis in pigs caused by the genus Serpulina, as well as to distinguish the species Serpulina hyodysenteriae and Serpulina pilosicoli in formalin......-fixed colon tissue sections. A genus-specific oligonucleotide probe SER1410 targeting the five species of porcine Serpulina was thus designed. Furthermore, species specific oligonucleotide probes (Hyo1210, Pilosi209 and Pilosi1405) were also designed to detect, identify and differentiate S. hyodysenteriae...

  7. Identification of Provocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

    Institute of Scientific and Technical Information of China (English)

    HOU Jianjun; LAI Hongyan; HUANG Bangqin; CHEN Jixin

    2009-01-01

    Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02>PM28S02>PM28S01>PM18S01,and that of the probes specific to T. pulchella was TP18S02>TP28S01>TP28S02>TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.

  8. Improved elucidation of biological processes linked to diabetic nephropathy by single probe-based microarray data analysis.

    Directory of Open Access Journals (Sweden)

    Clemens D Cohen

    Full Text Available BACKGROUND: Diabetic nephropathy (DN is a complex and chronic metabolic disease that evolves into a progressive fibrosing renal disorder. Effective transcriptomic profiling of slowly evolving disease processes such as DN can be problematic. The changes that occur are often subtle and can escape detection by conventional oligonucleotide DNA array analyses. METHODOLOGY/PRINCIPAL FINDINGS: We examined microdissected human renal tissue with or without DN using Affymetrix oligonucleotide microarrays (HG-U133A by standard Robust Multi-array Analysis (RMA. Subsequent gene ontology analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID showed limited detection of biological processes previously identified as central mechanisms in the development of DN (e.g. inflammation and angiogenesis. This apparent lack of sensitivity may be associated with the gene-oriented averaging of oligonucleotide probe signals, as this includes signals from cross-hybridizing probes and gene annotation that is based on out of date genomic data. We then examined the same CEL file data using a different methodology to determine how well it could correlate transcriptomic data with observed biology. ChipInspector (CI is based on single probe analysis and de novo gene annotation that bypasses probe set definitions. Both methods, RMA and CI, used at default settings yielded comparable numbers of differentially regulated genes. However, when verified by RT-PCR, the single probe based analysis demonstrated reduced background noise with enhanced sensitivity and fewer false positives. CONCLUSIONS/SIGNIFICANCE: Using a single probe based analysis approach with de novo gene annotation allowed an improved representation of the biological processes linked to the development and progression of DN. The improved analysis was exemplified by the detection of Wnt signaling pathway activation in DN, a process not previously reported to be involved in this disease.

  9. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    Science.gov (United States)

    Hu, Pan; Yang, Bin

    2016-01-15

    Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays.

  10. Positively charged polymer brush-functionalized filter paper for DNA sequence determination following Dot blot hybridization employing a pyrrolidinyl peptide nucleic acid probe.

    Science.gov (United States)

    Laopa, Praethong S; Vilaivan, Tirayut; Hoven, Voravee P

    2013-01-07

    As inspired by the Dot blot analysis, a well known technique in molecular biology and genetics for detecting biomolecules, a new paper-based platform for colorimetric detection of specific DNA sequences employing peptide nucleic acid (PNA) as a probe has been developed. In this particular study, a pyrrolidinyl PNA bearing a conformationally rigid d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was used as a probe. The filter paper was modified to be positively charged with grafted polymer brushes of quaternized poly(dimethylamino)ethyl methacrylate (QPDMAEMA) prepared by surface-initiated polymerization of 2-(dimethylamino)ethyl methacrylate from the filter paper via ARGET ATRP followed by quaternization with methyl iodide. Following the Dot blot format, a DNA target was first immobilized via electrostatic interactions between the positive charges of the QPDMAEMA brushes and negative charges of the phosphate backbone of DNA. Upon hybridization with the biotinylated pyrrolidinyl peptide nucleic acid (b-PNA) probe, the immobilized DNA can be detected by naked eye observation of the yellow product generated by the enzymatic reaction employing HRP-labeled streptavidin. It has been demonstrated that this newly developed assay was capable of discriminating between complementary and single base mismatch targets at a detection limit of at least 10 fmol. In addition, the QPDMAEMA-grafted filter paper exhibited a superior performance to the commercial membranes, namely Nylon 66 and nitrocellulose.

  11. 应用Alexa Fluor 488与BHQ1双标记寡核苷酸荧光探针测量辐射吸收剂量的可行性研究%The feasibility study of radiation absorbed dose measurement using oligonucleotide dually labeled with Alexa Fluor 488 and BHQ1 probe

    Institute of Scientific and Technical Information of China (English)

    林温文; 史盼影; 张保国

    2016-01-01

    目的 研究荧光探针Alexa Fluor 488-DNA-BHQ1用于辐射吸收剂量测量的可行性.方法 在寡核苷酸的5 '与3’端分别标记荧光分子Alexa Fluor 488与其特异性荧光抑制剂BHQ1,制备Alexa Fluor 488-DNA-BHQ1的DNA双标记荧光探针.用X射线照射其水溶液,检测照射后溶液的荧光强度.结果 浓度为0.5~1 μmol/L时,该荧光探针对剂量0.1~30 Gy之间线性响应最好(R2=0.99).在辐照后40~80 min检测探针,荧光强度基本不变.4℃条件下保存受照后的荧光探针的荧光稳定性较好.结论 荧光探针Alexa Fluor 488-DNA-BHQ1可以应用于0.1 ~30 Gy范围内辐射吸收剂量的测量.%Objective To study the feasibility of measuring radiation absorbed dose with the fluorescent probe Alexa Fluor 488-DNA-BHQ1.Methods An oligonucleotide dually labeled at its 5'-and 3'-end with fluorescent molecular Alexa Fluor 488 and specific fluorescence inhibitors BHQ1 was prepared.The Alexa Fluor 488-DNA-BHQ1 aqueous solution was exposed with X-ray and its fluorescence intensity was measured.Results When the concentration of Alexa Fluor 488-DNA-BHQ1 was between 0.5 and 1 μmol/L,the fluorescence intensity of its aqueous solution had excellent linear dose response from 0.1 to 30 Gy (R2 =0.99) and it was stably maintained after 40-80 min of irradiation especially at 4℃.Conclusions In the dose range of 0.1-30 Gy,the Alexa Fluor 488-DNA-BHQ1 fluorescent probe can be used to measure radiation absorbed dose.

  12. 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.

  13. Multiway study of hybridization in nanoscale semiconductor labeled DNA based on fluorescence resonance energy transfer

    DEFF Research Database (Denmark)

    Gholami, Somayeh; Kompany Zare, Mohsen

    2013-01-01

    The resolution of the ternary-binary complex competition of a target sequence and of its two complementary probes in sandwich DNA hybridization is reported. To achieve this goal, Fluorescence Resonance Energy Transfer (FRET) between oligonucleotide-functionalized quantum dot (QD) nanoprobes (QD...... in the photoluminescence excitation (PLE) plot. From the obtained data, energy transfer efficiency and Forster radius (R-0) were calculated. In particular, our results demonstrated that energy transfer by using QD donor-QD acceptor FRET pairs is more efficient in comparison with QD donor-organic dye acceptor pairs. Soft...

  14. Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable

    Science.gov (United States)

    Mirzabekov, Andrei; Guschin, Dmitry Y.; Chik, Valentine; Drobyshev, Aleksei; Fotin, Alexander; Yershov, Gennadiy; Lysov, Yuri

    2002-01-01

    This invention relates to using customized oligonucleotide microchips as biosensors for the detection and identification of nucleic acids specific for different genes, organisms and/or individuals in the environment, in food and in biological samples. The microchips are designed to convert multiple bits of genetic information into simpler patterns of signals that are interpreted as a unit. Because of an improved method of hybridizing oligonucleotides from samples to microchips, microchips are reusable and transportable. For field study, portable laser or bar code scanners are suitable.

  15. Adaptive resolution simulation of oligonucleotides

    Science.gov (United States)

    Netz, Paulo A.; Potestio, Raffaello; Kremer, Kurt

    2016-12-01

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  16. 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.

  17. Implementation and verification of a four-probe motion error measurement system for a large-scale roll lathe used in hybrid manufacturing

    Science.gov (United States)

    Chen, Yuan-Liu; Niu, Zengyuan; Matsuura, Daiki; Lee, Jung Chul; Shimizu, Yuki; Gao, Wei; Oh, Jeong Seok; Park, Chun Hong

    2017-10-01

    In this paper, a four-probe measurement system is implemented and verified for the carriage slide motion error measurement of a large-scale roll lathe used in hybrid manufacturing where a laser machining probe and a diamond cutting tool are placed on two sides of a roll workpiece for manufacturing. The motion error of the carriage slide of the roll lathe is composed of two straightness motion error components and two parallelism motion error components in the vertical and horizontal planes. Four displacement measurement probes, which are mounted on the carriage slide with respect to four opposing sides of the roll workpiece, are employed for the measurement. Firstly, based on the reversal technique, the four probes are moved by the carriage slide to scan the roll workpiece before and after a 180-degree rotation of the roll workpiece. Taking into consideration the fact that the machining accuracy of the lathe is influenced by not only the carriage slide motion error but also the gravity deformation of the large-scale roll workpiece due to its heavy weight, the vertical motion error is thus characterized relating to the deformed axis of the roll workpiece. The horizontal straightness motion error can also be synchronously obtained based on the reversal technique. In addition, based on an error separation algorithm, the vertical and horizontal parallelism motion error components are identified by scanning the rotating roll workpiece at the start and the end positions of the carriage slide, respectively. The feasibility and reliability of the proposed motion error measurement system are demonstrated by the experimental results and the measurement uncertainty analysis.

  18. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    Full Text Available A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH to evaluate accuracy of the results. We found that most (58.1% of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s, partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal

  19. A new route for local probing of inner interactions within a layered double hydroxide/benzene derivative hybrid material.

    Science.gov (United States)

    Fleutot, S; Dupin, J C; Baraille, I; Forano, C; Renaudin, G; Leroux, F; Gonbeau, D; Martinez, H

    2009-05-14

    This paper presents the preparation and characterization of hybrid hydrotalcite-type layered double hydroxides (Zn1-xAlx(OH)2HBSx.nH2O, with x=0.33) where HBS is the 4-phenol sulfonate, with a detailed analysis of the grafting process of this organic entity onto the host lattice. As a set of the usual techniques (XRD, TG-DT/MS, FTIR and 27Al MAS NMR) was used to characterize the hybrid materials, this work focuses on a joint study by X-ray photoelectron spectroscopy and some quantum-calculation modeling in order to highlight the nature of the interactions between the organic and the mineral sub-systems. For the as-prepared hybrid material, the main results lead to a quasi-vertical orientation of the organic molecules within the mineral sheets via H-bond stabilization. By heating the hybrid material up to 200 degrees C, the structure shrinks with the condensation of the organics; the different theoretical modeling done gives an energy-stable situation when a direct attachment of the HBS sulfonate group sets up with the mineral layers, in agreement with the recorded XPS experimental data.

  20. Probing the local environment of hybrid materials designed from ionic liquids and synthetic clay by Raman spectroscopy

    Science.gov (United States)

    Siqueira, Leonardo J. A.; Constantino, Vera R. L.; Camilo, Fernanda F.; Torresi, Roberto M.; Temperini, Marcia L. A.; Ribeiro, Mauro C. C.; Izumi, Celly M. S.

    2014-03-01

    Hybrid organic-inorganic material containing Laponite clay and ionic liquids forming cations have been prepared and characterized by FT-Raman spectroscopy, X-ray diffraction, and thermal analysis. The effect of varying the length of the alkyl side chain and conformations of cations has been investigated by using different ionic liquids based on piperidinium and imidazolium cations. The structure of the N,N-butyl-methyl-piperidinium cation and the assignment of its vibrational spectrum have been further elucidated by quantum chemistry calculations. The X-ray data indicate that the organic cations are intercalated parallel to the layers of the clay. Comparison of Raman spectra of pure ionic liquids with different anions and the resulting solid hybrid materials in which the organic cations have been intercalated into the clay characterizes the local environment experienced by the cations in the hybrid materials. The Raman spectra of hybrid materials suggest that the local environment of all confined cations, in spite of this diversity in properties, resembles the liquid state of ionic liquids with a relatively disordered structure.

  1. Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate.

    Science.gov (United States)

    Charles, Irudayasamy; Xi, Hongjuan; Arya, Dev P

    2007-01-01

    The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.

  2. Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labelled neurofilament cDNA probe.

    NARCIS (Netherlands)

    P. Liesi; J-P. Julien (Jean-Pierre); P. Vilja; F.G. Grosveld (Frank); L. Rechardt

    1986-01-01

    textabstractWe have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects t

  3. The Chemistry and Biology of Oligonucleotide Conjugates

    Science.gov (United States)

    Juliano, R.L.; Ming, Xin; Nakagawa, Osamu

    2012-01-01

    CONSPECTUS Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing they can interact with messenger mRNA or pre-mRNA targets with high selectivity and thus offer the possibility of precise manipulation of gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, with many candidates already in clinical trials. However, a major impediment to the maturation of oligonucleotide-based therapeutics is the fact that these relatively large and usually highly charged molecules have great difficulty crossing cellular membranes and thus in penetrating to their sites of action in the cytosol or nucleus. In this Account we first summarize some basic aspects of the biology of antisense and siRNA oligonucleotides and then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Our emphasis will be on the pharmacological ramifications of oligonucleotide conjugates rather than the details of conjugation chemistry. One important approach has been conjugation with ligands designed to bind to particular receptors and thus provide specificity to the interaction of cells with oligonucleotides. Another approach has been to couple antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Both of these approaches have enjoyed some success. However, there remains much to be learned before oligonucleotide conjugates can find an important place in human therapeutics. PMID:22353142

  4. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Mengjuan [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013 (China); Qian, Jing, E-mail: qianj@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg{sup 2+}. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg{sup 2+}, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg{sup 2+} in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg{sup 2+} contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg{sup 2+} quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg{sup 2+} detection. • The Hg{sup 2+} content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg{sup 2+} detection. • The ratiometric probe is of good simplicity, low toxicity, and

  5. Detection of iron-depositing Pedomicrobium species in native biofilms from the Odertal National Park by a new, specific FISH probe.

    Science.gov (United States)

    Braun, Burga; Richert, Inga; Szewzyk, Ulrich

    2009-10-01

    Iron-depositing bacteria play an important role in technical water systems (water wells, distribution systems) due to their intense deposition of iron oxides and resulting clogging effects. Pedomicrobium is known as iron- and manganese-oxidizing and accumulating bacterium. The ability to detect and quantify members of this species in biofilm communities is therefore desirable. In this study the fluorescence in situ hybridization (FISH) method was used to detect Pedomicrobium in iron and manganese incrusted biofilms. Based on comparative sequence analysis, we designed and evaluated a specific oligonucleotide probe (Pedo 1250) complementary to the hypervariable region 8 of the 16S rRNA gene for Pedomicrobium. Probe specificities were tested against 3 different strains of Pedomicrobium and Sphingobium yanoikuyae as non-target organism. Using optimized conditions the probe hybridized with all tested strains of Pedomicrobium with an efficiency of 80%. The non-target organism showed no hybridization signals. The new FISH probe was applied successfully for the in situ detection of Pedomicrobium in different native, iron-depositing biofilms. The hybridization results of native bioflims using probe Pedo_1250 agreed with the results of the morphological structure of Pedomicrobium bioflims based on scanning electron microscopy.

  6. Photochemical properties in flag leaves of a super-high-yielding hybrid rice and a traditional hybrid rice (Oryza sativa L.) probed by chlorophyll a fluorescence transient.

    Science.gov (United States)

    Zhang, Meiping; Shan, YongJie; Kochian, Leon; Strasser, Reto J; Chen, GuoXiang

    2015-12-01

    Chlorophyll a fluorescence of flag leaves in a super-high-yielding hybrid rice (Oryza sativa L.) LYPJ, and a traditional hybrid rice SY63 cultivar with lower grain yield, which were grown in the field, were investigated from emergence through senescence of flag leaves. As the flag leaf matured, there was an increasing trend in photosynthetic parameters such as quantum efficiency of primary photochemistry ([Formula: see text] Po) and efficiency of electron transport from PS II to PS I (Ψ Eo). The overall photosynthetic performance index (PIABS) was significantly higher in the high-yielding LYPJ compared to SY63 during the entire reproductive stage of the plant, the same to MDA content. However, [Formula: see text] Po(=F V/F M), an indicator of the primary photochemistry of the flag leaf, did not display significant changes with leaf age and was not significantly different between the two cultivars, suggesting that PIABS is a more sensitive parameter than [Formula: see text] Po (=F V/F M) during leaf age for distinguishing between cultivars differing in yield.

  7. In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition

    DEFF Research Database (Denmark)

    Kofoed, Michael Vedel; Stief, Peter; Poulsen, Morten

    In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition   Michael V.W. Kofoed, Peter Stief, Morten Poulsen, and Andreas Schramm Department of Biological Sciences, Microbiology, University of Aarhus, Denmark Denitrification, the sequential...... reductase) and nosZ (encoding nitrous oxide reductase), to detect nitrate-reducing and completely denitrifying bacteria, respectively. Enzyme-labelled oligonucleotide probes and digoxygenin-labelled polynucleotide probes were evaluated for in situ hybridization in combination with immunochemical detection...... reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying...

  8. Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents.

    Science.gov (United States)

    Shokrzadeh, Nasrin; Winkler, Anna-Maria; Dirin, Mehrdad; Winkler, Johannes

    2014-12-15

    Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG12-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2010-01-01

    Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

  10. Antisense oligonucleotides for the treatment of dyslipidaemia.

    Science.gov (United States)

    Visser, Maartje E; Witztum, Joseph L; Stroes, Erik S G; Kastelein, John J P

    2012-06-01

    Antisense oligonucleotides (ASOs) are short synthetic analogues of natural nucleic acids designed to specifically bind to a target messenger RNA (mRNA) by Watson-Crick hybridization, inducing selective degradation of the mRNA or prohibiting translation of the selected mRNA into protein. Antisense technology has the ability to inhibit unique targets with high specificity and can be used to inhibit synthesis of a wide range of proteins that could influence lipoprotein levels and other targets. A number of different classes of antisense agents are under development. To date, mipomersen, a 2'-O-methoxyethyl phosphorothioate 20-mer ASO, is the most advanced ASO in clinical development. It is a second-generation ASO developed to inhibit the synthesis of apolipoprotein B (apoB)-100 in the liver. In Phase 3 clinical trials, mipomersen has been shown to significantly reduce plasma low-density lipoprotein cholesterol (LDL-c) as well as other atherogenic apoB containing lipoproteins such as lipoprotein (a) [Lp(a)] and small-dense LDL particles. Although concerns have been raised because of an increase in intrahepatic triglyceride content, preliminary data from long-term studies suggest that with continued treatment, liver fat levels tend to stabilize or decline. Further studies are needed to evaluate potential clinical relevance of these changes. Proprotein convertase subtilisin/kexin-9 (PCSK9) is another promising novel target for lowering LDL-c by ASOs. Both second-generation ASOs and ASOs using locked nucleic acid technology have been developed to inhibit PCSK9 and are under clinical development. Other targets currently being addressed include apoC-III and apo(a) or Lp(a). By directly inhibiting the synthesis of specific proteins, ASO technology offers a promising new approach to influence the metabolism of lipids and to control lipoprotein levels. Its application to a wide variety of potential targets can be expected if these agents prove to be clinically safe and

  11. Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

    Directory of Open Access Journals (Sweden)

    Roberta D'Agata

    2017-01-01

    Full Text Available Gold nanoparticles (AuNPs exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

  12. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

    OpenAIRE

    1996-01-01

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model sys...

  13. Tuning molecular interactions in lipid-oligonucleotides assemblies via locked nucleic acid (LNA)-based lipids.

    Science.gov (United States)

    Patwa, Amit; Salgado, Gilmar; Dole, François; Navailles, Laurence; Barthélémy, Philippe

    2013-11-07

    Hybrid nucleotide-lipids containing locked nucleic acid (LNA) show enhanced hybridization properties with complementary single strand RNAs compared to DNA lipid analogues. The LNA adenosine lipid features unique binding properties with a high binding affinity for poly-uridine and the entropically driven formation of a stable complex (K(d) ≈ 43 nM). Enhanced hybridization properties of LNA-based lipids should be applicable for the development of oligonucleotide (ON) delivery systems or as small molecule binders to RNA for novel therapeutic strategies.

  14. Gene expression visualisation with antisense oligonucleotides; Visualisation de l'expression d'un gene: la strategie antisens

    Energy Technology Data Exchange (ETDEWEB)

    Brard, P.Y.; Gauchez, A.S.; Vuillez, J.P. [Universite Joseph-Fourier, Grenoble I, INSERM E 03-40, Radiopharmaceutiques Biocliniques, Faculte de Medecine, 38 (France); Defrancq, E. [Universite Joseph-Fourier, Grenoble I, UMR CNRS 5616 - LEDSS, Faculte de Medecine, 38 (France)

    2004-08-01

    Using radiolabelled antisense oligonucleotides to target mRNAs is a very promising method to study gene expression in vivo. This molecular imaging technique has the aim to identify cellular modifications in a very early stage of disease. During the last ten years, the number of published studies concerning in vivo tumor specific imaging is small. This fact depends on numerous biological challenges. In fact, gene specific oligonucleotides must be chemically modified to increase nuclease resistance and permit labelling with radionuclide. To be used as imaging radiopharmaceutical agent, a good antisense oligonucleotide need to valid a lot of steps: in vivo stability, cell membrane passage and durable hybridization to mRNA to obtain a kinetic which depends directly on gene expression level. We can get over these difficulties, we will illustrate with our experience on chemo-resistance imaging with antisense oligonucleotides which target h-mdr 1, in vitro and in vivo. (author)

  15. Limits on SUSY GUTs and Defects Formation in Hybrid Inflationary Models with Three-Year Wilkinson Microwave Anisotropy Probe (WMAP) Observations

    CERN Document Server

    Fraisse, A A

    2006-01-01

    We confront the predicted effects of hybrid inflationary models on the Cosmic Microwave Background (CMB) with three years of Wilkinson Microwave Anisotropy Probe (WMAP) observations. Using model selection, we compare the ability of a simple flat power-law LCDM model to describe the data to hybrid inflationary models involving global or local cosmic strings, or global textures. We find that it is statistically impossible to distinguish between these models: they all give a similar description of the data, the maximum ratio of the various evidences involved being never higher than e^{0.1 \\pm 0.5}. We then derive the maximum contribution that topological defects can make to the CMB, and place an upper bound on the possible value of cosmic strings tension of G\\mu \\leq 2.1 \\times 10^{-7} (68% CL). Finally, we give the corresponding constraints on the strings and D-strings masses, as well as limits on the D- and F-term coupling constants (\\kappa and \\lambda) and mass scales (M and \\sqrt{\\xi}).

  16. Frequencies of complex chromosome exchange aberrations induced by 238Pu alpha-particles and detected by fluorescence in situ hybridization using single chromosome-specific probes.

    Science.gov (United States)

    Griffin, C S; Marsden, S J; Stevens, D L; Simpson, P; Savage, J R

    1995-04-01

    We undertook an analysis of chromosome-type exchange aberrations induced by alpha-particles using fluorescence in situ hybridization (FISH) with whole chromosome-specific probes for human chromosomes 1 or 4, together with a pan-centromeric probe. Contact-inhibited primary human fibroblasts (in G1) were irradiated with 0.41-1.00 Gy 238Pu alpha-particles and aberrations were analysed at the next mitosis following a single chromosome paint. Exchange and aberration painting patterns were classified according to Savage and Simpson (1994a). Of exchange aberrations, 38-47% were found to be complex derived, i.e. resulting from three or more breaks in two or more chromosomes, and the variation with dose was minimal. The class of complex aberrations most frequently observed were insertions, derived from a minimum of three breaks in two chromosomes. There was also an elevated frequency of rings. The high level of complex aberrations observed after alpha-particle irradiation indicates that, when chromosome domains are traversed by high linear energy transfer alpha-particle tracks, there is an enhanced probability of production of multiple localized double-strand breaks leading to more complicated interactions.

  17. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    Science.gov (United States)

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  18. Injection site reactions after subcutaneous oligonucleotide therapy

    NARCIS (Netherlands)

    van Meer, L. (Leonie); M. Moerland (Matthijs); Gallagher, J. (Jolie); M.B.A. van Doorn (Martijn); E.P. Prens (Errol); A.F. Cohen; Rissmann, R. (Robert); J. Burggraaf (Jacobus)

    2016-01-01

    textabstractOligonucleotides (ONs) are short fragments of nucleic acids, currently being investigated as therapeutic agents. When administered subcutaneously (sc), ONs cause a specific local reaction originating around the injection site, such as erythema, itching, discomfort and pain, including

  19. 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)

  20. Signal enhancement for gene detection based on a redox reaction of [Fe(CN)(6)](4-) mediated by ferrocene at the terminal of a peptide nucleic acid as a probe with hybridization-amenable conformational flexibility.

    Science.gov (United States)

    Aoki, Hiroshi; Tao, Hiroaki

    2008-07-01

    Electrochemically enhanced DNA detection was demonstrated by utilizing the couple of a synthesized ferrocene-terminated peptide nucleic acid (PNA) with a cysteine anchor and a sacrificial electron donor [Fe(CN)(6)](4-). DNA detection sensors were prepared by modifying a gold electrode surface with a mixed monolayer of the probe PNA and 11-hydroxy-1-undecanethiol (11-HUT), protecting [Fe(CN)(6)](4-) from any unexpected redox reaction. Before hybridization, the terminal ferrocene moiety of the probe was subject to a redox reaction due to the flexible probe structure and, in the presence of [Fe(CN)(6)](4-), the observed current was amplified based on regeneration of the ferrocene moiety. Hybridization decreased the redox current of the ferrocene. This occurred because hybridization rigidified the probe structure: the ferrocene moiety was then removed from the electrode surface, and the redox reaction of [Fe(CN)(6)](4-) was again prevented. The change in the anodic current before and after hybridization was enhanced 1.75-fold by using the electron donor [Fe(CN)(6)](4-). Sequence-specific detection of the complementary target DNA was also demonstrated.

  1. A scanning probe-based pick-and-place procedure for assembly of integrated quantum optical hybrid devices

    CERN Document Server

    Schell, Andreas W; Schröder, Tim; Wolters, Janik; Aichele, Thomas; Benson, Oliver

    2011-01-01

    Integrated quantum optical hybrid devices consist of fundamental constituents such as single emitters and tailored photonic nanostructures. A reliable fabrication method requires the controlled deposition of active nanoparticles on arbitrary nanostructures with highest precision. Here, we describe an easily adaptable technique that employs picking and placing of nanoparticles with an atomic force microscope combined with a confocal setup. In this way, both the topography and the optical response can be monitored simultaneously before and after the assembly. The technique can be applied to arbitrary particles. Here, we focus on nanodiamonds containing single nitrogen vacancy centers, which are particularly interesting for quantum optical experiments on the single photon and single emitter level.

  2. Enumeration of respiring Pseudomonas spp. in milk within 6 hours by fluorescence in situ hybridization following formazan reduction.

    Science.gov (United States)

    Kitaguchi, Akiko; Yamaguchi, Nobuyasu; Nasu, Masao

    2005-05-01

    Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.

  3. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    Energy Technology Data Exchange (ETDEWEB)

    Fehm, Thomas Felix; Razansky, Daniel, E-mail: dr@tum.de [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany); Faculty of Medicine, Technische Universität München, Munich (Germany); Deán-Ben, Xosé Luís [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany)

    2014-10-27

    Ultrasonography and optoacoustic imaging share powerful advantages related to the natural aptitude for real-time image rendering with high resolution, the hand-held operation, and lack of ionizing radiation. The two methods also possess very different yet highly complementary advantages of the mechanical and optical contrast in living tissues. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition, and image reconstruction approaches. We report on a method for hybrid acquisition and reconstruction of three-dimensional pulse-echo ultrasound and optoacoustic images in real time based on passive ultrasound generation with an optical absorber, thus avoiding the hardware complexity of active ultrasound generation. In this way, complete hybrid datasets are generated with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at an unprecedented rate of 10 volumetric frames per second. Performance is subsequently showcased in phantom experiments and in-vivo measurements from a healthy human volunteer, confirming general clinical applicability of the method.

  4. A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Feng Zhou

    2015-09-01

    Full Text Available Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs as donors in luminescence resonance energy transfer (LRET. UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR excitation.

  5. Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    Science.gov (United States)

    Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

    2013-02-05

    A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

  6. Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

    Science.gov (United States)

    Ostromohov, Nadya; Schwartz, Ortal; Bercovici, Moran

    2015-09-15

    We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

  7. Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers.

    Science.gov (United States)

    Kaura, Mamta; Hrdlicka, Patrick J

    2015-07-14

    LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides. We hypothesized that the LNA monomers would alter the sugar rings of the modified DNA monomers toward more RNA-like North-type conformations for maximal DNA/RNA affinity and specificity. Indeed, the incorporation of LNA monomers almost invariably results in increased target affinity and specificity relative to the corresponding LNA-free ONs, but the magnitude of the stabilization varies greatly. Introduction of LNA nucleotides as direct neighbors into C5-pyrene-functionalized pyrimidine DNA monomers yields oligonucleotide probes with more desirable photophysical properties as compared to the corresponding LNA-free probes, including more intense fluorescence emission upon target binding and improved discrimination of single nucleotide polymorphisms (SNPs). These hybrid oligonucleotides are therefore promising probes for diagnostic applications.

  8. Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

    Directory of Open Access Journals (Sweden)

    K. Böhme

    2014-01-01

    Full Text Available Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB hybridization on membranes, coupled to the high specific ligation detection reaction (LDR. First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA. Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

  9. Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

    Science.gov (United States)

    Böhme, K.; Cremonesi, P.; Severgnini, M.; Villa, Tomás G.; Fernández-No, I. C.; Barros-Velázquez, J.; Castiglioni, B.; Calo-Mata, P.

    2014-01-01

    Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality. PMID:24818128

  10. Rapid detection of resistant genes of clinical isolates in Mycobatterium tuberculosis by membrane oligonucleotide probes hybridization%寡核苷酸探针膜杂交法检测耐药结核菌

    Institute of Scientific and Technical Information of China (English)

    梁建琴; 吴雪琼; 张俊仙; 陆阳; 李洪敏

    2006-01-01

    目的应用寡核苷酸探针膜杂交法快速检测临床分离株中结核分枝杆菌对异烟肼(INH)、链霉素(SM)、乙胺丁醇(EMB)的耐药性.方法设计与合成用于检测结核分枝杆菌3种药物常见耐药基因的寡核苷酸探针,点于硝酸纤维素膜上,与结核分枝杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交.结果 26株耐INH菌株中,16株Klb杂交阳性;7株inhla杂交阳性;23株耐SM菌株中,17株rpsl基因突变型探针Slb杂交阳性,2株突变型探针rrslb杂交阳性;31株耐EMB菌株中,13株Elb杂交阳性,1株Elc杂交阳性,5株Eld杂交阳性,1株Ele杂交阳性,11株与El探针杂交.kagG、inhA、rpsL、rrs和embB基因膜杂交突变检出率分别为61.5%,26.9%,74%,8.7%和64.5%,与PCR-DS分析结果一致.结论寡核苷酸探针膜杂交技术可能成为检测部分结核分枝杆菌耐药基因型简便、快速的方法.

  11. Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

    Energy Technology Data Exchange (ETDEWEB)

    Jafari, Safiye [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Faridbod, Farnoush, E-mail: faridbodf@khayam.ut.ac.ir [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Norouzi, Parviz [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Dezfuli, Amin Shiralizadeh [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Ajloo, Davood [School of Chemistry, Damghan University, Damghan (Iran, Islamic Republic of); Mohammadipanah, Fatemeh [Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 14155-6455 Tehran (Iran, Islamic Republic of); Ganjali, Mohammad Reza [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2015-10-01

    A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO{sub 2}NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy){sub 3}]{sup 2+/3+} redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy){sub 3}]{sup 2+/3+} FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10{sup −15} to 1 × 10{sup −8} mol L{sup −1}. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL{sup −1} with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy){sub 3}]{sup 2+/3+} interaction with ssDNA before and after hybridization. - Highlights: • New DNA biosensor is designed for sub-femtomolar detection of Aeromonas hydrophila DNA sequence. • Reduced graphene oxide decorated Ceria nanoparticles was used as a new immobilization platform. • Biosensor was successfully used to detect A. hydrophila DNA sequence in fish pond water.

  12. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection.

    Science.gov (United States)

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H; Brown, Tom

    2016-09-30

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis.

    Science.gov (United States)

    Chen, Qinghai; Wu, Nan; Xie, Meng; Zhang, Bo; Chen, Ming; Li, Jianjun; Zhuo, Lisha; Kuang, Hong; Fu, Weiling

    2012-04-01

    The accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations.

  14. Mapping genomic library clones using oligonucleotide arrays

    Energy Technology Data Exchange (ETDEWEB)

    Sapolsky, R.J.; Lipshutz, R.J. [Affymetrix, Santa Clara, CA (United States)

    1996-05-01

    We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adaptors. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density olligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection. 28 refs., 9 figs., 2 tabs.

  15. Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

    Directory of Open Access Journals (Sweden)

    Zonggao Shi

    2012-01-01

    Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

  16. Re-appraisal of the phylogeny and fluorescence in situ hybridization probes for the analysis of the Competibacteraceae in wastewater treatment systems

    DEFF Research Database (Denmark)

    McIlroy, Simon Jon; Nittami, Tadashi; Kanai, Eri

    2015-01-01

    to be of potential commercial interest for bioplastic production. In this study we have updated the 16S rRNA based phylogeny of the Competibacter- and the Plasticicumulans-lineages. The former is delineated by 13 clades including 2 described genera; “Ca. Competibacter” and “Ca. Contendobacter”. The oligonucleotide...

  17. Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Andrea Pauli

    Full Text Available Antisense oligonucleotides (ASOs are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

  18. A review of statistical methods for preprocessing oligonucleotide microarrays.

    Science.gov (United States)

    Wu, Zhijin

    2009-12-01

    Microarrays have become an indispensable tool in biomedical research. This powerful technology not only makes it possible to quantify a large number of nucleic acid molecules simultaneously, but also produces data with many sources of noise. A number of preprocessing steps are therefore necessary to convert the raw data, usually in the form of hybridisation images, to measures of biological meaning that can be used in further statistical analysis. Preprocessing of oligonucleotide arrays includes image processing, background adjustment, data normalisation/transformation and sometimes summarisation when multiple probes are used to target one genomic unit. In this article, we review the issues encountered in each preprocessing step and introduce the statistical models and methods in preprocessing.

  19. Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

    Science.gov (United States)

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Pertíñez, Samuel Núñez; Busto, Jon V; Martín, César; Alagia, Adele; Goñi, Félix M; Eritja, Ramón; Alkorta, Itziar

    2017-01-01

    The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    Science.gov (United States)

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  1. Development of a new deep-sea hybrid Raman insertion probe and its application to the geochemistry of hydrothermal vent and cold seep fluids

    Science.gov (United States)

    Zhang, Xin; Du, Zengfeng; Zheng, Ronger; Luan, Zhendong; Qi, Fujun; Cheng, Kai; Wang, Bing; Ye, Wangquan; Liu, Xiaorui; Lian, Chao; Chen, Changan; Guo, Jinjia; Li, Ying; Yan, Jun

    2017-05-01

    Hydrothermal vent fluids, cold seep fluids, their associated chemosynthetic communities, and the biogeochemical anaerobic oxidation of methane (AOM) play very important roles in the biogeochemical sulfur and carbon cycles in the ocean. Based on our previous success developing and deploying a deep-sea sediment pore water Raman probe, we developed a new deep-sea hybrid Raman insertion probe (RiP) designed to operate at temperatures up to 450 °C that can be inserted directly into high-temperature fluids emerging from hydrothermal vents. By routinely exchanging the various tips and optics of the probe, we can analyze the geochemistry of hydrothermal vent fluids, cold seep fluids, and sediment pore water profiles (0-60 cm) in situ. The instrument ensemble also includes a new deep-sea laser Raman spectrometer in a custom-designed, 6000-m titanium pressure housing, which is powered, controlled and deployed by the remotely operated vehicle (ROV) Faxian down to a maximum water depth of 4500 m. The new RiP was deployed at the Izena Hole hydrothermal area in the middle Okinawa Trough back-arc basin; the Papua-Australia-Canada-Manus (PACManus) hydrothermal vent area in the Manus back-arc basin, Papua New Guinea; and a cold seep field at Formosa Ridge in the northern South China Sea. The Raman peaks of CO2, CH4, H2S, HS-, SO42- and S8 were obtained in situ from high-temperature hydrothermal vents (290 °C), low-temperature cold seep fluids (2 °C) and the surrounding sediment pore water. Dissolved CH4 and S8 were identified for the first time in the fluids under the lush chemosynthetic communities of the cold seep. Several sediment pore water profiles collected near the cold seep were characterized by the loss of SO42- and increased CH4, H2S and HS- peaks. Additionally, the in situ pH range of the pore water profile was between 6.95 and 7.22. Thus, the RiP system provides a very useful tool for investigating the geochemistry of hydrothermal vent and cold seep fluids.

  2. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L. gene expression oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Paula Fernandez

    Full Text Available Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de. The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons. The resulting Sunflower Unigen Resource (SUR version 1.0 was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01 allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  3. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

    Science.gov (United States)

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  4. Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy

    Science.gov (United States)

    Lee, Jonathan R. I.; Bagge-Hansen, Michael; Tunuguntla, Ramya; Kim, Kyunghoon; Bangar, Mangesh; Willey, Trevor M.; Tran, Ich C.; Kilcoyne, David A.; Noy, Aleksandr; van Buuren, Tony

    2015-05-01

    Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. In this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ~30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural

  5. Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

    Science.gov (United States)

    Fontenete, Sílvia; Guimarães, Nuno; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-01-01

    The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.

  6. 用发夹寡核苷酸探针检测新生大鼠脑缺氧缺血时细胞凋亡的变化%Hairpin oligonucleotide probe as a marker of apoptotic change for cerebral hypoxia-ischemia in rat pups

    Institute of Scientific and Technical Information of China (English)

    朱长连; 王小阳; 杨平; 程秀永

    2001-01-01

    Aim: To investigate temporal and spatial changes of DNA double-stranded break (DSB) detected by biotin-labeled hairpin oligonucleotide probe(HPP) and compare with TdT-mediated dUTP nick end labeling (TUNEL) for detectiog DNA DSB in the immature rat brain after hypoxia-ischemia. Methods: 7-day-old rat pups were subjected to hypoxia-ischemia. The spatiotemporal pattern of DNA DSB was characterized with HPP in situ labeling. The relation of HPP labeling and brain damage was evaluated by double labeling with microtubule associated protein 2 (MAP 2). The sensitivity of HPP labeling was evaluated by double labeling with TUNEL.Results: HPP labeling localized in the ipsilateral hemisphere within the area of loss MAP 2 staining. The number of positive cells with nucleus condensation and cytoplasm shrinkage were remarkable with prolongation of reperfusion. The number of positive cells increased and reached peak at 24h post-HI. But in the nucleus habenularis displayed a faster time course. Hippocampus CA3, however, shows a more delayed time course. Double labeling of HPP and TUNEL showed more HPP labeling than TUNEL in the early reperfusion.Conclusion: HPP labeling shows typical apoptotic nucleus changes with different timepoints after HI. It is a sensitive marker for detecting apoptotic changes.%目的:观察用生物素标记的发夹寡核苷酸探针(HPP)检测未成熟大鼠脑缺氧缺血后DNA双链断裂(DSB)的短暂空间改变,并与TdT介导的dUTP缺口末端标记(TUNEL)的DNA DSB比较。方法:使7 d龄幼鼠缺氧缺血,用HPP位点标记显示DNA DSB短暂空间形式,用微管相关蛋白(MAP-2)双标记评价HPP标记和脑损伤的关系,用双标记的TUNEL评价HPP标记的灵敏度。结果:HPP标记局限在同侧半球MAP-2染色阴性区域;阳性细胞数与再灌注时间有关,在缺氧缺血后24 h,阳性细胞数上升达到高峰,但在松果体细胞核变化更早,而海马CA3区域细胞变化延迟。HPP和TUNEL双

  7. Lower hybrid frequency range waves generated by ion polarization drift due to electromagnetic ion cyclotron waves: Analysis of an event observed by the Van Allen Probe B

    Science.gov (United States)

    Khazanov, G. V.; Boardsen, S.; Krivorutsky, E. N.; Engebretson, M. J.; Sibeck, D.; Chen, S.; Breneman, A.

    2017-01-01

    We analyze a wave event that occurred near noon between 07:03 and 07:08 UT on 23 February 2014 detected by the Van Allen Probes B spacecraft, where waves in the lower hybrid frequency range (LHFR) and electromagnetic ion cyclotron (EMIC) waves are observed to be highly correlated, with Pearson correlation coefficient of 0.86. We assume that the correlation is the result of LHFR wave generation by the ions' polarization drift in the electric field of the EMIC waves. To check this assumption the drift velocities of electrons and H+, He+, and O+ ions in the measured EMIC wave electric field were modeled. Then the LHFR wave linear instantaneous growth rates for plasma with these changing drift velocities and different plasma compositions were calculated. The time distribution of these growth rates, their frequency distribution, and the frequency dependence of the ratio of the LHFR wave power spectral density (PSD) parallel and perpendicular to the ambient magnetic field to the total PSD were found. These characteristics of the growth rates were compared with the corresponding characteristics of the observed LHFR activity. Reasonable agreement between these features and the strong correlation between EMIC and LHFR energy densities support the assumption that the LHFR wave generation can be caused by the ions' polarization drift in the electric field of an EMIC wave.

  8. Hybridization Capture Using RAD Probes (hyRAD, a New Tool for Performing Genomic Analyses on Collection Specimens.

    Directory of Open Access Journals (Sweden)

    Tomasz Suchan

    Full Text Available In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD or performing size selection of the resulting fragments (in the case of single-digest RAD. Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD. In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites

  9. Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs

    DEFF Research Database (Denmark)

    Boye, Mette; Feenstra, Anne Avlund; Tegtmeier, Conny

    2000-01-01

    methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other...... closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S, suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated...... in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization...

  10. Detection of Lawsonia intracellularis in formalinfixed Porcine Intestinal Tissue Samples: Comparison of Immunofluorescense and In-situ Hybridization, and Evaluation of the Effectf of Controlled Autolysis

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Boesen, H. T.; Vigre, Håkan;

    2010-01-01

    Two methods, an immunofluorescence assay (IFA; with a Lawsonia intracellularis-specific monoclonal antibody) and fluorescent in-situ hybridization (FISH; with a specific oligonucleotide probe targeting 16S ribosomal RNA of the bacterium), were compared for their ability to detect L. intracellularis...... (the cause of porcine proliferative enteritis [PE]) in formalin-fixed samples of intestinal tissue. Of 69 intestinal samples with gross lesions of PE, 63 were positive by both FISH and IFA, but six were positive only by IFA. This indicated that the sensitivity of FISH was 91% that of IFA. However, both...

  11. Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

    Science.gov (United States)

    Harrison, J G; Liu, X; Balasubramanian, S

    1999-09-01

    A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.

  12. Electrochemical transduction of DNA hybridization at modified electrodes by using an electroactive pyridoacridone intercalator.

    Science.gov (United States)

    Bouffier, Laurent; Wang, Bingquan Stuart; Roget, André; Livache, Thierry; Demeunynck, Martine; Mailley, Pascal

    2014-02-01

    A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.

  13. Semi-automated, reverse-hybridization detection of multiple mutations causing hereditary fructose intolerance.

    Science.gov (United States)

    Kriegshäuser, Gernot; Halsall, David; Rauscher, Bettina; Oberkanins, Christian

    2007-06-01

    Hereditary fructose intolerance (HFI) is a potentially fatal nutritional disease that is caused by mutations in the liver isoenzyme of fructoaldolase (aldolase B). Our aim was to evaluate a diagnostic assay capable of simultaneously analyzing three-point mutations and a small deletion in the aldolase B (ALDOB) gene. The test under investigation is based on multiplex DNA amplification and hybridization to membrane strips presenting a parallel array of allele-specific oligonucleotide probes. We used the novel reverse-hybridization (RH) protocol to analyze 54 individuals previously genotyped by direct sequencing. RH genotyping for ALDOB mutations Delta4E4, A149P, A174D, and N334K was in complete concordance with results obtained by DNA sequencing. The procedure is rapid (<6h) and may be automated to a large extent. The RH assay tested in this study represents an accurate and robust screening tool to identify common ALDOB mutations.

  14. G-stack modulated probe intensities on expression arrays - sequence corrections and signal calibration

    Directory of Open Access Journals (Sweden)

    Fasold Mario

    2010-04-01

    Full Text Available Abstract Background The brightness of the probe spots on expression microarrays intends to measure the abundance of specific mRNA targets. Probes with runs of at least three guanines (G in their sequence show abnormal high intensities which reflect rather probe effects than target concentrations. This G-bias requires correction prior to downstream expression analysis. Results Longer runs of three or more consecutive G along the probe sequence and in particular triple degenerated G at its solution end ((GGG1-effect are associated with exceptionally large probe intensities on GeneChip expression arrays. This intensity bias is related to non-specific hybridization and affects both perfect match and mismatch probes. The (GGG1-effect tends to increase gradually for microarrays of later GeneChip generations. It was found for DNA/RNA as well as for DNA/DNA probe/target-hybridization chemistries. Amplification of sample RNA using T7-primers is associated with strong positive amplitudes of the G-bias whereas alternative amplification protocols using random primers give rise to much smaller and partly even negative amplitudes. We applied positional dependent sensitivity models to analyze the specifics of probe intensities in the context of all possible short sequence motifs of one to four adjacent nucleotides along the 25meric probe sequence. Most of the longer motifs are adequately described using a nearest-neighbor (NN model. In contrast, runs of degenerated guanines require explicit consideration of next nearest neighbors (GGG terms. Preprocessing methods such as vsn, RMA, dChip, MAS5 and gcRMA only insufficiently remove the G-bias from data. Conclusions Positional and motif dependent sensitivity models accounts for sequence effects of oligonucleotide probe intensities. We propose a positional dependent NN+GGG hybrid model to correct the intensity bias associated with probes containing poly-G motifs. It is implemented as a single-chip based calibration

  15. Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples.

    Science.gov (United States)

    Nasri, Soroush; Anjomshoaa, Ahmad; Song, Sarah; Guilford, Parry; McNoe, Les; Black, Michael; Phillips, Vicky; Reeve, Anthony; Humar, Bostjan

    2010-04-01

    Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

  16. On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

    Energy Technology Data Exchange (ETDEWEB)

    Noor, M. Omair; Tavares, Anthony J.; Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca

    2013-07-25

    Graphical abstract: -- Highlights: •Solid-phase multiplexed QD-FRET nucleic acid assay in electrokinetic fluidic chip. •Concurrent detection of two oligonucleotides based on channel length coverage. •Selection of “turn-on” and “turn-off” signals from two acceptor dyes and two colors of immobilized QDs, respectively. •No loss in assay sensitivity when implementing multiplexed assay format. -- Abstract: A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical

  17. In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi.

    Science.gov (United States)

    Velásquez, J N; Carnevale, S; Labbé, J H; Chertcoff, A; Cabrera, M G; Oelemann, W

    1999-01-01

    Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.

  18. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    Science.gov (United States)

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example.

  19. Methidium intercalator inserted into synthetic oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, E. N.; Smirnov, I. P.; Haff, L. A.; Tishchenko, E. I.; Mirzabekov, A. D.; Florentiev, V. L.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology; PerSeptive BioSystems Inc.

    1996-01-01

    A new methidium intercalator phosphoramidite has been synthesized. Methidium incorporation into an oligonucleotide during the synthesis was confirmed by UV and MALDI TOF MS data. UV melting experiments showed enhanced stability of a duplex, containing internal methidium. Methidium phosphoramidite has been synthesized and used for insertion of intercalator into the deoxyoligonucleotides.

  20. Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction.

    Science.gov (United States)

    González, Luis Miguel; Montero, Estrella; Sciutto, Edda; Harrison, Leslie J S; Parkhouse, R Michael E; Garate, Teresa

    2002-04-01

    The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.

  1. Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L.) reveals patterns of SNP variation associated with breeding.

    Science.gov (United States)

    Sim, Sung-Chur; Robbins, Matthew D; Chilcott, Charles; Zhu, Tong; Francis, David M

    2009-10-09

    Cultivated tomato (Solanum lycopersicum L.) has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP) discovery as a high-throughput approach for marker development in cultivated tomato. Three varieties, FL7600 (fresh-market), OH9242 (processing), and PI114490 (cherry) were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (alpha) used to define the confidence interval (CI), and ranged from 76% for polymorphisms identified at alpha or= 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of theta (Fst) suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace), and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka) to synonymous substitutions (Ks) for 20 loci with multiple SNPs (>or= 4 per locus). Six of 20 loci showed ratios of Ka/Ks >or= 0.9. Array-based SFP discovery was an efficient method to identify a large number of molecular markers for genetics and breeding in elite tomato germplasm. Patterns of sequence variation across five major tomato groups provided insight into to the effect of human selection on genetic variation.

  2. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  3. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    Directory of Open Access Journals (Sweden)

    Cheng-Chung Chou

    2012-12-01

    Full Text Available This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD-induced fluorescence resonance energy transfer (FRET reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor in a molar ratio of 10:1 (probe-to-QD, and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED, optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.

  4. Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

    Science.gov (United States)

    Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

    2014-04-01

    Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

  5. Development of an SH-SAW sensor for detection of DNA immobilization and hybridization

    Science.gov (United States)

    Roh, Yongrae; Woo, Jeongdong; Hur, Youngjune; Pak, Yukeun E.

    2005-05-01

    We have developed SH (shear horizontal) surface acoustic wave (SAW) sensors for detection of the immobilization and hybrdization of DNA (deoxyribonucleic acid) on the gold coated delay line of transverse SAW devices. The experiments of DNA immobilization and hybridization were performed with 15-mer oligonucleotides (probe and complementary target DNA). The sensor consists of twin SAW delay line oscillators (sensing channel and reference channel) operating at 100 MHz fabricated on 36° rotated Y-cut X-propagation LiTaO3 piezoelectric single crystals. The relative change in the frequency of the two oscillators was monitored to detect the immobilization of probe DNA with thiol group on the Au coated delay line and the hybridization between target DNA and immobilized probe DNA in a pH 7.4 PBS (phosphate buffered saline) solution. The measurement results showed a good response of the sensor to the mass loading effects of the DNA immobilization and hybridization with the sensitivity up to 1.5 ng/ml/Hz.

  6. Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

    Science.gov (United States)

    Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

    2010-01-01

    Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

  7. Tailoring bifunctional hybrid organic–inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu2+ ions

    Science.gov (United States)

    Tomina, Veronika V; Melnyk, Inna V; Zub, Yuriy L; Kareiva, Aivaras; Vaclavikova, Miroslava; Kessler, Vadim G

    2017-01-01

    Spherical silica particles with bifunctional (≡Si(CH2)3NH2/≡SiCH3, ≡Si(CH2)3NH2/≡Si(CH2)2(CF2)5CF3) surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and 13C and 29Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR) and electron spectroscopy of diffuse reflectance (ESDR) spectroscopy using Cu2+ ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl) groups improves the kinetic characteristics of the samples during the sorption of copper(II) ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl) groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature. PMID:28243572

  8. Tailoring bifunctional hybrid organic-inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu(2+) ions.

    Science.gov (United States)

    Tomina, Veronika V; Melnyk, Inna V; Zub, Yuriy L; Kareiva, Aivaras; Vaclavikova, Miroslava; Seisenbaeva, Gulaim A; Kessler, Vadim G

    2017-01-01

    Spherical silica particles with bifunctional (≡Si(CH2)3NH2/≡SiCH3, ≡Si(CH2)3NH2/≡Si(CH2)2(CF2)5CF3) surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and (13)C and (29)Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR) and electron spectroscopy of diffuse reflectance (ESDR) spectroscopy using Cu(2+) ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl) groups improves the kinetic characteristics of the samples during the sorption of copper(II) ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl) groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature.

  9. Tailoring bifunctional hybrid organic–inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu2+ ions

    Directory of Open Access Journals (Sweden)

    Veronika V. Tomina

    2017-02-01

    Full Text Available Spherical silica particles with bifunctional (≡Si(CH23NH2/≡SiCH3, ≡Si(CH23NH2/≡Si(CH22(CF25CF3 surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT spectroscopy, and 13C and 29Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR and electron spectroscopy of diffuse reflectance (ESDR spectroscopy using Cu2+ ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl groups improves the kinetic characteristics of the samples during the sorption of copper(II ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature.

  10. Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.

    Science.gov (United States)

    Cano, R J; Palomares, J C; Torres, M J; Klem, R E

    1992-07-01

    This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.

  11. Generation of chicken Z-chromosome painting probes by microdissection for screening large-insert genomic libraries.

    Science.gov (United States)

    Zimmer, R; King, W A; Verrinder Gibbins, A M

    1997-01-01

    A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10-15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200-800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.

  12. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  13. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  14. Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells.

    Science.gov (United States)

    Politz, J C; Taneja, K L; Singer, R H

    1995-01-01

    Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells. Images PMID:8559650

  15. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  16. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles.

    Science.gov (United States)

    Ita, Kevin

    2017-03-01

    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Ultrahigh molecular recognition specificity of competing DNA oligonucleotide strands in thermal equilibrium

    CERN Document Server

    Schenkelberger, Marc; Mai, Timo; Ott, Albrecht

    2016-01-01

    The specificity of molecular recognition is important to molecular self-organization. A prominent example is the biological cell where, within a highly crowded molecular environment, a myriad of different molecular receptor pairs recognize their binding partner with astonishing accuracy. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide strand can prevail against a disproportionally more concentrated competitor that exhibits reduced affinity due to a mismatch. The magnitude of deviation from the simple picture above may reach several orders of magnitude. In our experiments the effective molecular affinity of a given strand remains...

  18. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    Science.gov (United States)

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation. Copyright © 2015. Published by Elsevier B.V.

  19. A novel modification of real-time AS-qPCR by using locked nucleic acid-modified oligonucleotide probe as wild type allele amplification blockers for quantitative detection of the JAK2 V617F mutation%评价AS-LNA-qPCR法检测JAK2 V617F突变率的临床应用价值

    Institute of Scientific and Technical Information of China (English)

    邵冬华; 梁国威; 何美琳; 曹清芸

    2013-01-01

    Objective To develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.Methods Based on the real-time allele-specific PCR (AS-qPCR),the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR,and then a novel AS-LNA-qPCR method was established.The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values.We validated intra-and inter-assay variability for quantifying JAK2 V617F.We also assayed 623 apparent healthy donors by our method to validate its clinical application value.Results The quantitative lower limit of this method for JAK2 V617F was 0.01%,and the intra-and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%,respectively.Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors,and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.Conclusion The AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.%目的 建立一种定量检测外周血细胞酪氨酸激酶2(JAK2)基因V617F突变率的等位基因特异性实时荧光定量PCR(AS-qPCR)方法.方法 在AS-qPCR基础上,引入1条锁核酸(LNA)修饰的寡核苷酸探针,用以选择性抑制AS-qPCR中突变引物对野生等位基因的非特异性扩增,定量检测JAK2 V617F突变率,称之为AS-LNA-qPCR法.通过AS-LNA-qPCR法测定样本的循环阈值(Ct值),根据AS-LNA-qPCR法检测不同JAK2 V617F突变率标准品的Ct值,绘制标准曲线,根据标准曲线直接获得检测样本中JAK2 V617F突变率.

  20. 流式细胞仪结合序列特异性寡核苷酸探针基因分型方法在血小板供者资料库建立中的应用%Genotyping by Flow-Sequence Specific Oligonucleotide Probes to Establish a Human Platelet Donor Database

    Institute of Scientific and Technical Information of China (English)

    周丹; 庄乃宝; 孔令魁

    2012-01-01

    目的 探讨高通量流式细胞仪结合序列特异性寡核苷酸探针(FLOW-SSO)技术在人类血小板抗原( HPA)基因分型中的应用.方法 采用FLOW-SSO技术对1378例汉族血小板捐献者HPA1~6,15系统的基因型进行检测;并以10份国际输血协会(ISBT)第12届血小板免疫学术研讨会提供的质控样品对FLOW-SSO技术的可靠性进行考核.以3家供应商提供的不同聚合酶链反应-序列特异引物法(PCR-SSP)试剂,对50份血小板样品的基因分型结果进行比对试验.分型结果采用x2检验考察HPA系统的基因型频率分布是否符合Hardy-Weinberg遗传平衡定律.结果 通过FLOW-SSO方法获得的1379例血小板供者HPA等位基因频率如下,HPA-1a为0.9927,HPA-1b为0.0073,HPA-2a为0.9536,HPA-2b为0.0464,HPA-3a为0.5700,HPA-3b为0.4300,HPA-4a为0.9982,HPA -4b为0.0018,HPA-5a为0.9804,HPA-5b为0.0196,HPA-6a为0.9822,HPA-6b为0.0178,HPA- 15a为0.5337及HPA-15b为0.4663.基因频率检测结果经x2检验,其分布符合Hardy-Weinberg遗传平衡定律.ISBT提供的10份样品考核结果相符率为100%.50份血小板样本采用3种不同的PCR-SSP试剂测定的HPA1~6,15基因分型结果,与FLOW-SSO技术测定结果完全一致.结论 FLOW-SSO基因分型技术有便捷、重复性好及高通量等特点,适用于血小板供者资料库的血小板基因分型.%Objective To explore the application of the high-throughput flow-sequence specific oligonucleotide probes (FLOW-SSO) technology in human platelet antigen (HPA) genotyping. Method A total of 1378 platelet donors of Han ethnic group were genotyped by FLOW-SSO for HPA system 1-6 and 15 Ten reference samples provided from 12th platelet immunology workshop of International Society of Blood Transfusion (ISBT) were tested as control group, and 50 samples were detected by the PCR-SSP with 3 different manufacturing sources reagents as the comparison test. x2 test was used to study the genotype frequencies of the HPA system

  1. Mesoscale hybrid calibration artifact

    Science.gov (United States)

    Tran, Hy D.; Claudet, Andre A.; Oliver, Andrew D.

    2010-09-07

    A mesoscale calibration artifact, also called a hybrid artifact, suitable for hybrid dimensional measurement and the method for make the artifact. The hybrid artifact has structural characteristics that make it suitable for dimensional measurement in both vision-based systems and touch-probe-based systems. The hybrid artifact employs the intersection of bulk-micromachined planes to fabricate edges that are sharp to the nanometer level and intersecting planes with crystal-lattice-defined angles.

  2. Electrochemical study of hepta–oligonucleotides

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    Zdenka Balcarova

    2010-12-01

    Full Text Available The study deals with the description and characterization of twohepta–oligonucleotides (DNA and RNA forming special structures.We studied their electrochemical behaviour by means of cyclicvoltammetry (CV and elimination voltammetry with linear scan(EVLS in combination with adsorptive stripping (AdS technique.Differences in electrochemical behaviour of hepta–deoxyribonucleotide and its RNA analog were discussed with regardto their different structures in solutions and their melting points.

  3. Abundant oligonucleotides common to most bacteria.

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    Colin F Davenport

    Full Text Available BACKGROUND: Bacteria show a bias in their genomic oligonucleotide composition far beyond that dictated by G+C content. Patterns of over- and underrepresented oligonucleotides carry a phylogenetic signal and are thus diagnostic for individual species. Patterns of short oligomers have been investigated by multiple groups in large numbers of bacteria genomes. However, global distributions of the most highly overrepresented mid-sized oligomers have not been assessed across all prokaryotes to date. We surveyed overrepresented mid-length oligomers across all prokaryotes and normalised for base composition and embedded oligomers using zero and second order Markov models. PRINCIPAL FINDINGS: Here we report a presumably ancient set of oligomers conserved and overrepresented in nearly all branches of prokaryotic life, including Archaea. These oligomers are either adenine rich homopurines with one to three guanine nucleosides, or homopyridimines with one to four cytosine nucleosides. They do not show a consistent preference for coding or non-coding regions or aggregate in any coding frame, implying a role in DNA structure and as polypeptide binding sites. Structural parameters indicate these oligonucleotides to be an extreme and rigid form of B-DNA prone to forming triple stranded helices under common physiological conditions. Moreover, the narrow minor grooves of these structures are recognised by DNA binding and nucleoid associated proteins such as HU. CONCLUSION: Homopurine and homopyrimidine oligomers exhibit distinct and unusual structural features and are present at high copy number in nearly all prokaryotic lineages. This fact suggests a non-neutral role of these oligonucleotides for bacterial genome organization that has been maintained throughout evolution.

  4. Surface modification of poly(dimethylsiloxane) (PDMS) microchannels with DNA capture-probes for potential use in microfluidic DNA analysis systems

    Science.gov (United States)

    Khodakov, Dmitriy A.; Thredgold, Leigh D.; Lenehan, Claire E.; Andersson, Gunther A.; Kobus, Hilton; Ellis, Amanda V.

    2011-12-01

    Poly(dimethylsiloxane) (PDMS) is an elastomeric material used for microfluidic devices and is especially suited to medical and forensic applications. This is due to its relatively low cost, ease of fabrication, excellent optical transmission characteristics and its ability to support electroosmotic flow, required during electrophoretic separations. These aspects combined with its large range of surface modification chemistries, make PDMS an attractive substrate in microfluidic devices for, in particular, DNA separation. Here, we report the successful wet chemical surface modification of PDMS microchannels using a simple three step method to produce an isothiocyanate-terminated surface. Initially, PDMS was oxygen plasma treated to produce a silanol-terminated surface, this was then reacted with 3-aminopropyltriethoxysilane with subsequent reaction of the now amine-terminated surface with p-phenylenediisothiocyanate. Water contact angle measurements both before and after modification showed a reduction in hydrophobicity from 101o for native PDMS to 94o for the isothiocyante-terminated PDMS. The isothiocyanate-terminated surface was then coupled with an amineterminated single-stranded DNA (ssDNA) oligonucleotide capture probe via a thiourea linkage. Confirmation of capture probe attachment was observed using fluorescent microscopy after hybridization of the capture probes with fluorescently labeled complimentary ssDNA oligonucleotides.

  5. Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

    Science.gov (United States)

    Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

    2012-01-01

    Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

  6. Competitive reporter monitored amplification (CMA--quantification of molecular targets by real time monitoring of competitive reporter hybridization.

    Directory of Open Access Journals (Sweden)

    Thomas Ullrich

    Full Text Available BACKGROUND: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. METHODOLOGY AND PRINCIPAL FINDINGS: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. CONCLUSIONS AND SIGNIFICANCE: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2, we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls

  7. 共有序列简并杂合寡核苷酸引物聚合酶链反应在呼吸道副黏病毒科病毒诊断中的应用%Consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction-A powerful technique for the identification of Paramyxoviridae in clinical specimens

    Institute of Scientific and Technical Information of China (English)

    赵百慧; 张泓; 张曦; 王春; 沈佳仁; 俞雪莲; 高烨; 滕峥; 朱兆奎; 储维; 宋黎黎

    2013-01-01

    The present paper aims to compare the sensitivity, specificity and accordance rate of consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction (CODEHOP PCR) technique and commercial kit in diagnosis of paramyxovirus infection in clinical specimens and to further evaluate the value of CODEHOP PCR assay in detection of known and novel respiratory viruses . A total of 572 specimens from lower respiratory tract were collected from Children' Hospital of Shanghai inpatients with acute respiratory infections during 2011. These specimens were analyzed by CODEHOP PCR and commercial RV12 kit for the detection of paramyxoviruses, including known parainfluenza virus type 1 (PIV-1), PIV-2 , PIV-3, respiratory syncytial virus A (RSVA) , RSVB , human metapneumovirus (HMPV) and novel viruses . The results showed that 102 out of 572 specimens (19 .76% ) were detected positive by the RV12 kit, and 113 were detected positive by CODEHOP PCR . The accordance rate of the two methods was 85.39% . CODEHOP PCR is more sensitive than RV12 kit in detecting RSVA infection in cell culture . The study suggests that CODEHOP PCR is a powerful technique for the identification of paramyxoviruses in clinical specimens . It can be optimized by combining with some new techniques , and is worthy of application in the future.%采用共有序列简并杂合寡核苷酸引物聚合酶链反应(CODEHOP PCR)体系和商品化RV12试剂盒对急性呼吸道感染患儿下呼吸道标本中的副黏病毒科病毒进行检测,比较CODEHOP PCR与RV12试剂盒检测结果的符合率和敏感度,观察CODEHOP PCR在临床呼吸道标本中对副黏病毒诊断的应用价值,进一步探讨具备检测已知呼吸道病毒和未知新病毒特点的CODEHOP PCR在呼吸道疾病谱和未知潜在呼吸道病毒诊断中的推广价值.采集2011年上海市儿童医院因急性呼吸道感染住院的患儿下呼吸道标本572份,分别采用2种方法

  8. DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.

    Science.gov (United States)

    Brückner, Kathrin; Schwarz, Kathleen; Beck, Sebastian; Linscheid, Michael W

    2014-01-07

    The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.

  9. Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization

    Directory of Open Access Journals (Sweden)

    Chetta M

    2008-01-01

    Full Text Available Background: Standard methods of mutation detection are time consuming in Hemophilia A (HA rendering their application unavailable in some analysis such as prenatal diagnosis. Objectives: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH as an alternative and reliable tool for mutation detection in FVIII gene. Patients/Methods: We have applied a new method of cSBH that uses two different colors for detection of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously characterized individuals with FVIII deficiency. Results: Data show that, when solution-phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be successfully detected. The technique was reliable also in identifying a mutant FVIII allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr in exon 16 and three novel neutral polymorphisms are presented with an updated protocol for 2-color cSBH. Conclusions: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients.

  10. Quantitative fluorescent in-situ hybridization: a hypothesized competition mode between two dominant bacteria groups in hydrogen-producing anaerobic sludge processes.

    Science.gov (United States)

    Huang, C-L; Chen, C-C; Lin, C-Y; Liu, W-T

    2009-01-01

    Two hydrogen-producing continuous flow stirred tank reactors (CSTRs) fed respectively with glucose and sucrose were investigated by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH). The substrate was fed in a continuous mode decreased from hydraulic retention time (HRT) 10 hours to 6, 5, 4, 3, and 2 hours. Quantitative fluorescent in-situ hybridization (FISH) observations further demonstrated that two morphotypes of bacteria dominated both microbial communities. One was long rod bacteria which can be targeted either by Chis150 probe designed to hybridize the gram positive low G + C bacteria or the specific oligonucleotide probe Lg10-6. The probe Lg10-6, affiliated with Clostridium pasteurianum, was designed and then checked with other reference organisms. The other type, unknown group, which cannot be detected by Chis150 was curved rod bacteria. Notably, the population ratios of the two predominant groups reflected the different operational performance of the two reactors, such as hydrogen producing rates, substrate turnover rates and metabolites compositions. Therefore, a competition mode of the two dominant bacteria groups was hypothesized. In the study, 16S rRNA-based gene library of hydrogen-producing microbial communities was established. The efficiency of hydrogen yields was correlated with substrates (glucose or sucrose), HRT, metabolites compositions (acetate, propionate, butyrate and ethanol), thermal pre-treatment (seed biomass was heated at 100 degrees C for 45 minutes), and microbial communities in the bioreactor, not sludge sources (municipal sewage sludge, alcohol-processing sludge, or bean-processing sludge). The designed specific oligonucleotide probe Lg10-6 also provides us a useful and fast molecular tool to screen hydrogen-producing microbial communities in the future research.

  11. Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

    Science.gov (United States)

    Danaher, Patrick; White, Robin Lynn; Hanson, Erin K; Ballantyne, Jack

    2015-01-01

    A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required

  12. A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides.

    Science.gov (United States)

    Magner, Dorota; Biala, Ewa; Lisowiec-Wachnicka, Jolanta; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

  13. Template-Directed Ligation of Peptides to Oligonucleotides

    Science.gov (United States)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  14. Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus.

    Science.gov (United States)

    Dong, Shibiao; Zhao, Rongtao; Zhu, Jiangong; Lu, Xiao; Li, Yang; Qiu, Shaofu; Jia, Leili; Jiao, Xiong; Song, Shiping; Fan, Chunhai; Hao, RongZhang; Song, HongBin

    2015-04-29

    A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3',5,5'-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens.

  15. Fluorescence in situ hybridization rapidly detects three different pathogenic bacteria in urinary tract infection samples.

    Science.gov (United States)

    Wu, Qing; Li, Yan; Wang, Ming; Pan, Xiao P; Tang, Yong F

    2010-11-01

    The detection of pathogenic bacteria in urine is an important criterion for diagnosing urinary tract infections (UTIs). By using fluorescence in situ hybridization (FISH) with rRNA-targeted, fluorescently labeled oligonucleotide probes, bacterial pathogens present in urine samples were identified within 3-4 h. In this study, three probes that are specific for Escherichia coli, Enterococcus faecalis and Staphylococcus aureus were designed based on the conserved 16S RNA sequences, whereas probe Eub338 broadly recognizes all bacteria. We collected a total of 1000 urine samples, and 325 of these samples tested positive for a UTI via traditional culturing techniques; additionally, all 325 of these samples tested positive with the Eub338 probe in FISH analysis. FISH analyses with species-specific probes were performed in parallel to the test the ability to differentiate among several pathogenic bacteria. The samples for these experiments included 76 E. coli infected samples, 32 E. faecalis infected samples and 9 S. aureus infected samples. Compared to conventional methods of bacterial identification, the FISH method produced positive results for >90% of the samples tested. FISH has the potential to become an extremely useful diagnostic tool for UTIs because it has a quick turnaround time and high accuracy.

  16. The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry

    Science.gov (United States)

    Adams, Nicholas M.; Olmsted, Ian R.; Haselton, Frederick R.; Bornhop, Darryl J.; Wright, David W.

    2013-01-01

    Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼105 target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits. PMID:23519610

  17. Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.

    Science.gov (United States)

    Bekker, Cornelis P J; de Vos, Sander; Taoufik, Amar; Sparagano, Olivier A E; Jongejan, Frans

    2002-10-22

    The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.

  18. Guanine-tethered antisense oligonucleotides as synthetic riboregulators.

    Science.gov (United States)

    Hagihara, Masaki

    2014-01-01

    Regulation of gene expression by short oligonucleotides (antisense oligonucleotides), which can modulate RNA structures and inhibit subsequent associations with the translation machinery, is a potential approach for gene therapy. This chapter describes an alternative antisense strategy using guanine-tethered antisense oligonucleotides (G-ASs) to introduce a DNA-RNA heteroquadruplex structure at a designated sequence on RNA targets. The feasibility of using G-ASs to modulate RNA conformation may allow control of RNA function by inducing biologically important quadruplex structures. This approach to manipulate quadruplex structures using G-ASs may expand the strategies for regulating RNA structures and the functions of short oligonucleotide riboregulators.

  19. Improved anchorage of Ti6Al4V orthopaedic bone implants through oligonucleotide mediated immobilization of BMP-2 in osteoporotic rats.

    Directory of Open Access Journals (Sweden)

    Julia V Wölfle

    Full Text Available The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.

  20. Improved Anchorage of Ti6Al4V Orthopaedic Bone Implants through Oligonucleotide Mediated Immobilization of BMP-2 in Osteoporotic Rats

    Science.gov (United States)

    Wölfle, Julia V.; Fiedler, Jörg; Dürselen, Lutz; Reichert, Judith; Scharnweber, Dieter; Förster, Anne; Schwenzer, Bernd; Reichel, Heiko; Ignatius, Anita; Brenner, Rolf E.

    2014-01-01

    The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo. PMID:24465929

  1. A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces.

    Science.gov (United States)

    Halliwell, C M; Cass, A E

    2001-06-01

    The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.

  2. New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE) of Y chromosome mixtures.

    Science.gov (United States)

    Rothe, Jessica; Watkins, Norman E; Nagy, Marion

    2012-01-01

    Allele-specific extension reactions (ASERs) use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

  3. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2013-01-01

    Mobile probing is a method, developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time and space......). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings point...... to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

  4. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  5. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  6. Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge.

    Science.gov (United States)

    Olszewska, Magdalena A; Kocot, Aleksandra M; Nynca, Anna; Łaniewska-Trokenheim, Łucja

    2016-11-01

    pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.

  7. The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Leffers, H

    1993-01-01

    We describe a method for screening of cDNA libraries with highly degenerate oligonucleotides using tetramethylammonium chloride (TMAC). This method is a convenient alternative to using probes generated by the polymerase chain reaction (PCR), especially when these cannot easily be made. Nylon filt...

  8. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Chen Feng

    2010-10-01

    Full Text Available Abstract Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals and Plasmodium falciparum (a related parasite responsible for severe human malaria, we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at

  9. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeu