WorldWideScience

Sample records for hybrid pathogen dna

  1. Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA.

    Science.gov (United States)

    Walter, Anne; Wu, Jie; Flechsig, Gerd-Uwe; Haake, David A; Wang, Joseph

    2011-03-09

    An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Hybrid Pathogen DNA Detector:Users? Manual v1.5

    Energy Technology Data Exchange (ETDEWEB)

    Schikora, B; Hietala, S; Shi, L; Lee, L; Skowronski, E; Ardans, A

    2004-01-12

    The Hybrid Unit uses an advanced fluidic design to move very small reagent samples through many unit operations to complete complex molecular biology experiments. The primary use of this machine is to analyze a small liquid sample for the highly specific presence of select agents known to be used in bio-warfare. The Hybrid Unit is coupled with a Luminex bead detection unit for the multiplexing of many assays in one tube. Because of this, multiple controls can be included in each run to avoid false positives. The built-in flow through PCR unit amplifies specific DNA signatures and increases sensitivity, thereby limiting exposure of handlers to highly concentrated (and potentially hazardous, spore containing) sample volumes. The reproducible precision of the Hybrid Unit also gives confidence when a signal is given that detects an agent in a given sample.

  3. A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

    Science.gov (United States)

    Hailemariam, Zerihun; Ahmed, Jabbar Sabir; Clausen, Peter-Henning; Nijhof, Ard Menzo

    2017-01-01

    An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic(®) cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex(®) resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  5. DNA chip based sensor for amperometric detection of infectious pathogens.

    Science.gov (United States)

    Singh, Swati; Kaushal, Ankur; Khare, Shashi; Kumar, Ashok

    2017-10-01

    Several infectious pathogens are found in human whose detection is essential for rapid cure of diseases. The most commonly found pathogen in human is Streptococcus pyogenes which leads to a wide range of infections from mild pharyngitis to rheumatic heart disease. An ultrasensitive DNA chip based sensor was developed for quick identification of pathogen S. pyogenes from patient throat swab samples. The amperometric response was measured after hybridization of specific probe with single stranded genomic DNA (ssG-DNA) from the patient samples. The DNA chip was characterized by FTIR, SEM and validated with suspected patient real samples. The sensitivity of the DNA chip based sensor was found 951.34(μA/cm(2))/ng DNA and lower limit of detection (LOD) was 130fg/6μL samples. The DNA chip based sensor is highly specific and takes only 30min for identification of specific pathogen. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. DNA-based hybrid catalysis.

    Science.gov (United States)

    Rioz-Martínez, Ana; Roelfes, Gerard

    2015-04-01

    In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed.

  7. Hybridization and Selective Release of DNA Microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J; Benett, W; Mukerjee, E; Dzenitis, J; Wheeler, E K

    2011-11-29

    DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy

  8. DNA microarray technique for detecting food-borne pathogens

    Directory of Open Access Journals (Sweden)

    Xing GAO

    2012-08-01

    Full Text Available Objective To study the application of DNA microarray technique for screening and identifying multiple food-borne pathogens. Methods The oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogens, and then were validated by bioinformatic analyses. The 5' end of each probe was modified by amino-group and 10 Poly-T, and the optimized probes were synthesized and spotted on aldehyde-coated slides. The bacteria DNA template incubated with Klenow enzyme was amplified by arbitrarily primed PCR, and PCR products incorporated into Aminoallyl-dUTP were coupled with fluorescent dye. After hybridization of the purified PCR products with DNA microarray, the hybridization image and fluorescence intensity analysis was acquired by ScanArray and GenePix Pro 5.1 software. A series of detection conditions such as arbitrarily primed PCR and microarray hybridization were optimized. The specificity of this approach was evaluated by 16 different bacteria DNA, and the sensitivity and reproducibility were verified by 4 food-borne pathogens DNA. The samples of multiple bacteria DNA and simulated water samples of Shigella dysenteriae were detected. Results Nine different food-borne bacteria were successfully discriminated under the same condition. The sensitivity of genomic DNA was 102 -103pg/ μl, and the coefficient of variation (CV of the reproducibility of assay was less than 15%. The corresponding specific hybridization maps of the multiple bacteria DNA samples were obtained, and the detection limit of simulated water sample of Shigella dysenteriae was 3.54×105cfu/ml. Conclusions The DNA microarray detection system based on arbitrarily primed PCR can be employed for effective detection of multiple food-borne pathogens, and this assay may offer a new method for high-throughput platform for detecting bacteria.

  9. Detecting hybridization using ancient DNA.

    Science.gov (United States)

    Schaefer, Nathan K; Shapiro, Beth; Green, Richard E

    2016-06-01

    It is well established that related species hybridize and that this can have varied but significant effects on speciation and environmental adaptation. It should therefore come as no surprise that hybridization is not limited to species that are alive today. In the last several decades, advances in technologies for recovering and sequencing DNA from fossil remains have enabled the assembly of high-coverage genome sequences for a growing diversity of organisms, including many that are extinct. Thanks to the development of new statistical approaches for detecting and quantifying admixture from genomic data, genomes from extinct populations have proven useful both in revealing previously unknown hybridization events and informing the study of hybridization between living organisms. Here, we review some of the key recent statistical innovations for detecting ancient hybridization using genomewide sequence data and discuss how these innovations have revised our understanding of human evolutionary history.

  10. The inheritance of pathogenic mitochondrial DNA mutations

    OpenAIRE

    Cree, L.M.; Samuels, D.C.; Chinnery, P F

    2009-01-01

    Abstract Mitochondrial DNA mutations cause disease in >1 in 5000 of the population, and ~1 in 200 of the population are asymptomatic carriers of a pathogenic mtDNA mutation. Many patients with these pathogenic mtDNA mutations present with a progressive, disabling neurological syndrome that leads to major disability and premature death. There is currently no effective treatment for mitochondrial disorders, placing great emphasis on preventing the transmission of these diseases. An e...

  11. Large scale multiplex PCR improves pathogen detection by DNA microarrays

    Directory of Open Access Journals (Sweden)

    Krönke Martin

    2009-01-01

    Full Text Available Abstract Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. Conclusion Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

  12. DNA hybridization on silicon nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shalini, E-mail: shalinsin@gmail.co [Electronic Materials Division, National Physical Laboratory (CSIR), Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Faculty of Life Science, Aligarh Muslim University, Aligarh-202001 (India); Zack, Jyoti [Dr. B.R Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007 (India); Kumar, Dinesh; Srivastava, S.K.; Govind [Electronic Materials Division, National Physical Laboratory (CSIR), Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Saluja, Daman [Dr. B.R Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007 (India); Khan, M.A. [Faculty of Life Science, Aligarh Muslim University, Aligarh-202001 (India); Singh, P.K. [Electronic Materials Division, National Physical Laboratory (CSIR), Dr. K. S. Krishnan Marg, New Delhi-110012 (India)

    2010-11-30

    Nanowire-based detection strategies provide promising new routes to bioanalysis and indeed are attractive to conventional systems because of their small size, high surface-to-volume ratios, electronic, and optical properties. A sequence-specific detection of single-stranded oligonucleotides using silicon nanowires (SiNWs) is demonstrated. The surface of the SiNWs is functionalized with densely packed organic monolayer via hydrosilylation for covalent attachment. Subsequently, deoxyribonucleic acid (DNA) is immobilized to recognize the complementary target DNA. The biomolecular recognition properties of the nanowires are tested via hybridization with {sup {gamma}P32} tagged complementary and non-complementary DNA oligonucleotides, showing good selectivity and reversibility. No significant non-specific binding to the incorrect sequences is observed. X-ray photoelectron spectroscopy, fluorescence imaging, and nanodrop techniques are used to characterize the modified SiNWs and covalent attachment with DNA. The results show that SiNWs are excellent substrates for the absorption, stabilization and detection of DNA sequences and could be used for DNA microarrays and micro fabricated SiNWs DNA sensors.

  13. Microcantilver-based DNA hybridization sensors for Salmonella identification

    Directory of Open Access Journals (Sweden)

    Carlo Ricciardi

    2012-02-01

    Full Text Available The detection of pathogenic microorganisms in foods remains a challenging since the safety of foodstuffs has to be ensured by the food producing companies. Conventional methods for the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Biomolecular methods, are commonly used as a support for traditional techniques, thanks to their high sensitivity, specificity and not excessive costs. However, new methods like biosensors for example, can be an exciting alternative to the more traditional tecniques for the detection of pathogens in food. In this study we report Salmonella enterica serotype Enteritidis DNA detection through a novel class of label-free biosensors: microcantilevers (MCs. In general, MCs can operate as a microbalance and is used to detect the mass of the entities anchored to the cantilever surface using the decrease in the resonant frequency. We use DNA hybridization as model reaction system and for this reason, specific single stranded probe DNA of the pathogen and three different DNA targets (single-stranded complementary DNA, PCR product and serial dilutions of DNA extracted from S. Enteritidis strains were applied. Two protocols were reported in order to allow the probe immobilization on cantilever surface: i MC surface was functionalized with 3-aminopropyltriethoxysilane and glutaraldehyde and an amino-modified DNA probe was used; ii gold-coated sensors and thiolated DNA probes were used in order to generate a covalent bonding (Th-Au. For the first one, measures after hybridization with the PCR product showed related frequency shift 10 times higher than hybridization with complementary probe and detectable signals were obtained at the concentrations of 103 and 106 cfu/mL after hybridization with bacterial DNA. There are currently optimizations of the second protocol, where preliminary results have shown to be more uniform and therefore more precise within each of the

  14. Phytophthora x pelgrandis, a new natural hybrid pathogenic to Pelargonium grandiflorum hort.

    Science.gov (United States)

    Nirenberg, Helgard I; Gerlach, Wolfram F; Gräfenhan, Tom

    2009-01-01

    A new Phytophthora hybrid of Ph. cactorum (Leb. & Cohn) Schroet. and Ph. nicotianaevan Breda de Haan pathogenic to cultivars of Pelargonium grandiflorum hort. is described as Phytophthora X pelgrandis and its morphological features are documented. Morphological, physiological (e.g., temperature requirements) and molecular data (DNA sequencing, random amplified polymorphic DNA-PCR) are presented for isolates of the Phytophthora hybrid. Pathogenicity was tested on cultivars of P. grandiflorum and Nicotiana tabacum. For comparison cultures of the parental species and additional Phytophthora taxa also were examined.

  15. Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

    Science.gov (United States)

    Tymoczko, Jakub; Schuhmann, Wolfgang; Gebala, Magdalena

    2014-12-24

    Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

  16. Kinetics and dynamics of DNA hybridization.

    Science.gov (United States)

    Yin, Yandong; Zhao, Xin Sheng

    2011-11-15

    DNA hybridization, wherein strands of DNA form duplex or larger hybrids through noncovalent, sequence-specific interactions, is one of the most fundamental processes in biology. Developing a better understanding of the kinetic and dynamic properties of DNA hybridization will thus help in the elucidation of molecular mechanisms involved in numerous biochemical processes. Moreover, because DNA hybridization has been widely adapted in biotechnology, its study is invaluable to the development of a range of commercially important processes. In this Account, we examine recent studies of the kinetics and dynamics of DNA hybridization, including (i) intramolecular collision of random coil, single-stranded DNA (ssDNA), (ii) nucleic acid hairpin folding, and (iii) considerations of DNA hybridization from both a global view and a detailed base-by-base view. We also examine the spontaneous single-base-pair flipping in duplex DNA because of its importance to both DNA hybridization and repair. Intramolecular collision of random coil ssDNA, with chemical relaxation times ranging from hundreds of nanoseconds to a few microseconds, is investigated both theoretically and experimentally. The first passage time theory of Szabo, Schulten, and Schulten, which determines the average reaction time of the intrachain collision, was tested. Although it was found to provide an acceptable approximation, a more sophisticated theoretical treatment is desirable. Nucleic acid hairpin folding has been extensively investigated as an important model system of DNA hybridization. The relaxation time of hairpin folding and unfolding strongly depends on the stem length, and it may range from hundreds of microseconds to hundreds of milliseconds. The traditional two-state model has been revised to a multistate model as a result of new experimental observations and theoretical study, and partially folded intermediate states have been introduced to the folding energy landscape. On the other hand, new

  17. Mechanisms of Surface-Mediated DNA Hybridization

    Science.gov (United States)

    2015-01-01

    Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100 000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state. PMID:24708278

  18. Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization.

    Science.gov (United States)

    do Nascimento, Cássio; Muller, Katia; Sato, Sandra; Albuquerque Junior, Rubens Ferreira

    2012-04-01

    Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at -20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p  0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.

  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. Clinical strains of acinetobacter classified by DNA-DNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Tjernberg, I.; Ursing, J. (Department of Medical Microbiology, University of Lund, Malmoe General Hospital, Malmoe (Sweden))

    1989-01-01

    A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author).

  1. Control of DNA hybridization by photoswitchable molecular glue.

    Science.gov (United States)

    Dohno, Chikara; Nakatani, Kazuhiko

    2011-12-01

    Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

  2. Hybridization characteristics of biomolecular adaptors, covalent DNA streptavidin conjugates

    NARCIS (Netherlands)

    Niemeyer, CM; Burger, W; Hoedemakers, RMJ

    1998-01-01

    Semisynthetic, covalent streptavidin-DNA adducts are versatile molecular connectors for the fabrication of both nano-and microstructured protein arrays by use of DNA hybridization. In this study, the hybridization characteristics of six adduct species, each containing a different DNA sequence of 21

  3. DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA

    DEFF Research Database (Denmark)

    Christensen, H.; Angen, Øystein; Mutters, R.;

    2000-01-01

    by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance...

  4. Control of aggregation-induced emission by DNA hybridization

    OpenAIRE

    Li, Shaoguang; Langenegger, Simon Matthias; Häner, Robert

    2013-01-01

    Aggregation-induced emission (AIE) was studied by hybridization of dialkynyl-tetraphenylethylene (DATPE) modified DNA strands. Molecular aggregation and fluorescence of DATPEs are controlled by duplex formation.

  5. Bioelectronic DNA detection of human papillomaviruses using eSensor™: a model system for detection of multiple pathogens

    Directory of Open Access Journals (Sweden)

    Miller Donna L

    2003-06-01

    Full Text Available Abstract Background We used human papillomaviruses (HPV as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™ in identifying multiple pathogens. Methods Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. Results Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. Conclusions This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens.

  6. Hybrid male sterility is caused by mitochondrial DNA deletion.

    Science.gov (United States)

    Hayashida, Kenji; Kohno, Shigeru

    2009-07-01

    Although it is known that the hybrid male mouse is sterile just like any other animal's heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.

  7. In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

    Science.gov (United States)

    Lermo, A; Campoy, S; Barbé, J; Hernández, S; Alegret, S; Pividori, M I

    2007-04-15

    A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

  8. IDENTIFICATION OF PATHOGENIC LEPTOSPIRES BY RECOMBINANT DNA PROBES

    Institute of Scientific and Technical Information of China (English)

    戴保民; 肖建国; 沈成义

    1994-01-01

    Early diagnosis of leptospirosis of pulmonary diffuse bernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific method for diagnvsis, a genonlic library of the main pathogen of PDH, L. interogans serovar lai strath 017, was constructed with the plasmid vector pUC9. Recmbinant plasmids which have hornologotLq fragments of pathogenic inptospires were screened from the bank. A recombinant plasmid.designated pCX7, could detect 1. 7 kb fragment of strain 017. 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdo-maclis, respectively, without cross hybridization with nonpathogcnic leptospires such as L. biflexa strain Patoc 1 and Leptonema illini. The recombinant plasmid pCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.

  9. [Detection of DNA human cytomegalovirus of a molecular methods: hybrid capture DNA CMV by immunocompromised].

    Science.gov (United States)

    Mhiri, Leila; Arrouji, Zakia; Slim, Amine; Ben Redjeb, Saida

    2006-10-01

    Human cytomegalovirus (HCMV), a member of the beta-virus herpes family, is a ubiquitous human pathogen. After a primary infection, HCMV establishes life latency. HCMV rarely causes symptomatic disease in an immunocompetent host, however, it is a major cause of infectious morbidity and mortality in immunocompromised individuals and developing fetuses. The HCMV genome consists of 240 kbp of double stranded DNA. Early diagnosis molecular of CMV infection is important. The objective of this study was to develop a molecular methods: Quantitative Hybrid capture for the detection of DNA CMV. We present results for 200 immunocompromised collected from 1999 to 2003 (122 men and 78 women, whom mean age was 35 years). Our results showed that 25% of women and 36% of men were positif for hybrid capture DNA CMV. This simple test (cold probe) provide quantitative and fast results. Also the efficacity of anti-CMV therapy can be followed. More over, in contrary with pp65-antigenemia assay and CMV PCR, this test can be managed on biopsy sample.

  10. Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation.

    Science.gov (United States)

    Zhao, Likun; Zhang, Jiangyan; Xu, Huiming; Geng, Hao; Cheng, Yongqiang

    2016-09-01

    Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity.

  11. [DNA-DNA hybridization in several species of Hansenula].

    Science.gov (United States)

    Poncet, S; Fiol, J B; Billon-Grand, G

    1984-05-30

    The genus Hansenula was considered a long time ago as a good pattern for phylogenetic research. In 1969, Wickerham proposed an evolutive scheme based upon morphological, physiological and ecological criteria. Recently, relatedness among yeasts were analysed by DNA-DNA hybridization in liquid medium. H. anomala var. anomala (G + C content: 37.1%) was compared with H. anomala var. schneggii (37.6%), H. subpelliculosa (33.8%) line 3, H. sydowiorum (40.1%) and H. muscicola (37.1%). These results showed little relatedness between H. anomala var. anomala/H. ciferrii and H. anomala var. anomala/H. subpelliculosa. On the other hand, H. anomala var. schneggii shared 89.5% of its nucleotide sequences with H. anomala var. anomala. These 2 strains were considered to represent the same species. H. holstii showed 67.1% complementarity with H. anomala var. anomala: this strain is considered to represent valid species, different from H. anomala var. anomala, but H. muscicola with 72.5% relatedness to H. anomala var. anomala could be considered as a 'limit species'. An unexpected finding was that H. beckii was closely related to H. anomala var. anomala (84.8%). These data suggested the inadequacy of current criteria used to establish the phylogenetic lines in genus Hansenula.

  12. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  13. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    Science.gov (United States)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  14. A liquid-crystal-based DNA biosensor for pathogen detection

    Science.gov (United States)

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-03-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection.

  15. Crowding-induced Cooperativity in DNA Surface Hybridization

    Science.gov (United States)

    Lei, Qun-li; Ren, Chun-lai; Su, Xiao-hang; Ma, Yu-qiang

    2015-01-01

    High density DNA brush is not only used to model cellular crowding, but also has a wide application in DNA-functionalized materials. Experiments have shown complicated cooperative hybridization/melting phenomena in these systems, raising the question that how molecular crowding influences DNA hybridization. In this work, a theoretical modeling including all possible inter and intramolecular interactions, as well as molecular details for different species, is proposed. We find that molecular crowding can lead to two distinct cooperative behaviours: negatively cooperative hybridization marked by a broader transition width, and positively cooperative hybridization with a sharper transition, well reconciling the experimental findings. Moreover, a phase transition as a result of positive cooperativity is also found. Our study provides new insights in crowding and compartmentation in cell, and has the potential value in controlling surface morphologies of DNA functionalized nano-particles. PMID:25875056

  16. Food Microbial Pathogen Detection and Analysis Using DNA Microarray Technologies

    OpenAIRE

    Rasooly, Avraham; Herold, Keith E.

    2008-01-01

    Culture-based methods used for microbial detection and identification are simple to use, relatively inexpensive, and sensitive. However, culture-based methods are too time-consuming for high-throughput testing and too tedious for analysis of samples with multiple organisms and provide little clinical information regarding the pathogen (e.g., antibiotic resistance genes, virulence factors, or strain subtype). DNA-based methods, such as polymerase chain reaction (PCR), overcome some these limit...

  17. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    Science.gov (United States)

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  18. Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.; Fang, Ferric C.

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

  19. Specific hydrolysis of DNA by Ce4+-ODN hybrid

    Institute of Scientific and Technical Information of China (English)

    SHEN; Hebai; ZHOU; Wenjun; YANG; Yongtao; ZHANG; Feng

    2004-01-01

    The cerium complex bound to the derivative of oligoDNA has been synthesized successfully that can hydrolyze DNA with sequence-specificity. The synthesized derivative of oligoDNA, 5′-EDTA-P-10 mers ODN, was demonstrated by HPLC. The fluorescence spectrum of Tb3+ was detected after its interaction with the hybrid of 10-mers ODN and 26-mers ODN and the results show that the artificial endo-enzyme can recognize and combine firmly with the substrate DNA. The electrophorogram shows that the cerium-oligoDNA hybrid can specifically hydrolyze its substrate DNA and the cleavage site of this hydrolysis reaction is also discussed. This artificial nuclease can be widely used in molecular biology and genetic engineering as one kind of endo-enzyme.

  20. DNA transfer in the gastric pathogen Helicobacter pylori.

    Science.gov (United States)

    Fernandez-Gonzalez, Esther; Backert, Steffen

    2014-04-01

    The gastric pathogen Helicobacter pylori is one of the most genetically diverse bacteria. Recombination and DNA transfer contribute to its genetic variability and enhance host adaptation. Among the strategies described to increase genetic diversity in bacteria, DNA transfer by conjugation is one of the best characterized. Using this mechanism, a fragment of DNA from a donor cell can be transferred to a recipient, always mediated by a conjugative nucleoprotein complex, which is evolutionarily related to type IV secretion systems (T4SSs). Interestingly, the H. pylori chromosomes can encode up to four T4SSs, including the cagPAI, comB, tfs3, and tfs4 genes, some of which are known to promote chronic H. pylori infection. The T4SS encoded by the cagPAI mediates the injection of the effector protein CagA and proinflammatory signaling, and the comB system is involved in DNA uptake from the environment. However, the role of tfs3 and tfs4 is not yet clear. The presence of a functional XerD tyrosine recombinase and 5'AAAGAATG-3' border sequences as well as two putative conjugative relaxases (Rlx1 and Rlx2), a coupling protein (TraG), and a chromosomal region carrying a putative origin of transfer (oriT) suggest the existence of a DNA transfer apparatus in tfs4. Moreover, a conjugation-like DNA transfer mechanism in H. pylori has already been described in vitro, but whether this occurs in vivo is still unknown. Some extrachromosomal plasmids and phages are also present in various H. pylori strains. Genetic exchange among plasmids and chromosomes, and involved DNA mobilization events, could explain part of H. pylori's genetic diversity. Here, we review our knowledge about the possible DNA transfer mechanisms in H. pylori and its implications in bacterial adaptation to the host environment.

  1. The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range.

    Directory of Open Access Journals (Sweden)

    Patrik Inderbitzin

    Full Text Available Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages.

  2. Specific DNA-RNA Hybrid Recognition by TAL Effectors

    Directory of Open Access Journals (Sweden)

    Ping Yin

    2012-10-01

    Full Text Available The transcription activator-like (TAL effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats. Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.

  3. DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

    Science.gov (United States)

    Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

    2017-01-11

    DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

  4. Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis.

    Science.gov (United States)

    Bangun, A; Johnson, J L; Tripathy, D N

    1987-01-01

    DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.

  5. DNA hybridization and ligation for directed colloidal assembly

    Science.gov (United States)

    Shyr, Margaret

    Colloidal assembly using DNA hybridization has been pursued as a means assemble non-conventional ordered colloidal structures. However, to date it is undetermined whether DNA hybridization can be used to achieve non-FCC colloidal crystals. Using microcontact printing techniques, we have fabricated covalently bound single stranded DNA (ssDNA) two-dimensional arrays on glass surfaces, which were used to direct the assembly of complementary DNA functionalized polystyrene colloids. Two of the hallmarks of DNA hybridization, sequence specificity and thermal reversibility, were demonstrated. Due to the periodicity of these arrays, laser diffraction was used to directly monitor these structures during assembly. To demonstrate the versatility of the 2D colloidal array assembled via DNA hybridization, a catalytic DNA sequence or DNAzyme was incorporated into the colloidal array system. By tethering the enzymatic strand to the patterned glass surface and the substrate strand to polystyrene colloids, we showed that the DNAzyme could prevent the assembly of the arrays when the required Pb2+ cofactor was provided. Attempts to assemble the colloid arrays and disassemble via the Pb2+-DNAzyme induced cleavage were unsuccessful, likely due to the incomplete cleavage of the multitude of hybridized linkages between each colloid and the surface. Since DNA is not only capable of catalyzing reactions, but also capable of being reacted upon by a variety of biological enzymes, we examined the use of DNA ligase as a means to control the assembly of DNA-functionalized colloids. A three-sequence linker system was used for the hybridization mediated assembly of colloids: one sequence was tethered to the surface of the glass slide or colloids, one was tethered to another colloid surface, and the linker sequence hybridizes simultaneously to both tethered sequences. Once hybridized, the two tethered fragments can be ligated using DNA ligase, resulting in a continuous sequence tethered on one end

  6. Cytogenetic analysis from DNA by comparative genomic hybridization.

    Science.gov (United States)

    Tachdjian, G; Aboura, A; Lapierre, J M; Viguié, F

    2000-01-01

    Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.

  7. DNA Polymer Brush Patterning through Photocontrollable Surface-Initiated DNA Hybridization Chain Reaction.

    Science.gov (United States)

    Huang, Fujian; Zhou, Xiang; Yao, Dongbao; Xiao, Shiyan; Liang, Haojun

    2015-11-18

    The fabrication of DNA polymer brushes with spatial resolution onto a solid surface is a crucial step for biochip research and related applications, cell-free gene expression study, and even artificial cell fabrication. Here, for the first time, a DNA polymer brush patterning method is reported based on the photoactivation of an ortho-nitrobenzyl linker-embedded DNA hairpin structure and a subsequent surface-initiated DNA hybridization chain reaction (HCR). Inert DNA hairpins are exposed to ultraviolet light irradiation to generate DNA duplexes with two active sticky ends (toeholds) in a programmable manner. These activated DNA duplexes can initiate DNA HCR to generate multifunctional patterned DNA polymer brushes with complex geometrical shapes. Different multifunctional DNA polymer brush patterns can be fabricated on certain areas of the same solid surface using this method. Moreover, the patterned DNA brush surface can be used to capture target molecules in a desired manner.

  8. Instructing cells with programmable peptide DNA hybrids

    Science.gov (United States)

    Freeman, Ronit; Stephanopoulos, Nicholas; Álvarez, Zaida; Lewis, Jacob A.; Sur, Shantanu; Serrano, Chris M.; Boekhoven, Job; Lee, Sungsoo S.; Stupp, Samuel I.

    2017-07-01

    The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed.

  9. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    Science.gov (United States)

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  10. DNA hybridization catalysts and catalyst circuits

    OpenAIRE

    SEELIG, Georg; Yurke, Bernard; Winfree, Erik

    2005-01-01

    Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that carry out their functions through the catalysis of metastable fuels into waste products. Catalytic control of reaction rates will prove to be as useful and ubiquitous in DNA nanotechnology as it is in biology. Here we present experimental results on the control of the decay rates of a metastable DNA "fuel". We show that the fuel complex can be induced to decay with a r...

  11. Oligo-DNA custom macroarray for monitoring major pathogenic and non-pathogenic fungi and bacteria in the phyllosphere of apple trees.

    Directory of Open Access Journals (Sweden)

    Ying-Hong He

    Full Text Available BACKGROUND: To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees. METHODS AND FINDINGS: First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 10(3 CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants. CONCLUSIONS: The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in

  12. Absence of pathogenic mitochondrial DNA mutations in mouse brain tumors

    Directory of Open Access Journals (Sweden)

    Seyfried Thomas N

    2005-08-01

    Full Text Available Abstract Background Somatic mutations in the mitochondrial genome occur in numerous tumor types including brain tumors. These mutations are generally found in the hypervariable regions I and II of the displacement loop and unlikely alter mitochondrial function. Two hypervariable regions of mononucleotide repeats occur in the mouse mitochondrial genome, i.e., the origin of replication of the light strand (OL and the Arg tRNA. Methods In this study we examined the entire mitochondrial genome in a series of chemically induced brain tumors in the C57BL/6J strain and spontaneous brain tumors in the VM mouse strain. The tumor mtDNA was compared to that of mtDNA in brain mitochondrial populations from the corresponding syngeneic mouse host strain. Results Direct sequencing revealed a few homoplasmic base pair insertions, deletions, and substitutions in the tumor cells mainly in regions of mononucleotide repeats. A heteroplasmic mutation in the 16srRNA gene was detected in a spontaneous metastatic VM brain tumor. Conclusion None of the mutations were considered pathogenic, indicating that mtDNA somatic mutations do not likely contribute to the initiation or progression of these diverse mouse brain tumors.

  13. Isothermal hybridization kinetics of DNA assembly of two-dimensional DNA origami.

    Science.gov (United States)

    Song, Jie; Zhang, Zhao; Zhang, Shuai; Liu, Lei; Li, Qiang; Xie, Erqing; Gothelf, Kurt Vesterager; Besenbacher, Flemming; Dong, Mingdong

    2013-09-09

    The Watson-Crick base-pairing with specificity and predictability makes DNA molecules suitable for building versatile nanoscale structures and devices, and the DNA origami method enables researchers to incorporate more complexities into DNA-based devices. Thermally controlled atomic force microscopy in combination with nanomechanical spectroscopy with forces controlled in the pico Newton (pN) range as a novel technique is introduced to directly investigate the kinetics of multistrand DNA hybridization events on DNA origami nanopores under defined isothermal conditions. For the synthesis of DNA nanostructures under isothermal conditions at 60 °C, a higher hybridization rate, fewer defects, and a higher stability are achieved compared to room-temperature studies. By quantifying the assembly times for filling pores in origami structures at several constant temperatures, the fill factors show a consistent exponential increase over time. Furthermore, the local hybridization rate can be accelerated by adding a higher concentration of the staples. The new insight gained on the kinetics of staple-scaffold hybridization on the synthesis of two dimensional DNA origami structures may open up new routes and ideas for designing DNA assembly systems with increased potential for their application.

  14. Effects of humic substances on fluorometric DNA quantification and DNA hybridization

    NARCIS (Netherlands)

    Bachoon, DS; Otero, E; Hodson, RE

    2001-01-01

    DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities, Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic

  15. DNA meets synthetic polymers—highly versatile hybrid materials

    NARCIS (Netherlands)

    Alemdaroglu, Fikri E.; Herrmann, Andreas

    2007-01-01

    The combination of synthetic polymers and DNA has provided biologists, chemists and materials scientists with a fascinating new hybrid material. The challenges in preparing these molecular chimeras were overcome by different synthetic strategies that rely on coupling the nucleic acid moiety and the

  16. Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

    Directory of Open Access Journals (Sweden)

    Md. Mahbubur Rahman

    2015-02-01

    Full Text Available Conducting polymers (CPs are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective.

  17. In vitro assembly of multiple DNA fragments using successive hybridization.

    Science.gov (United States)

    Jiang, Xinglin; Yang, Jianming; Zhang, Haibo; Zou, Huibin; Wang, Cong; Xian, Mo

    2012-01-01

    Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

  18. FY02 CBNP Annual Report: Discovery of DNA Signature of Biothreat Detection Using Suppression Subtractive Hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, G L; Radnedge, L

    2002-11-19

    Our goal is to develop robust DNA signatures for rapid and specific DNA-based detection platforms that can be employed by CBNP to detect a wide range of potential agents. Our approach has resulted in highly specific DNA signatures for Yersina pestis, Bacillus anthracis and Brucella species. Furthermore, this approach can be applied to any genome (even uncharacterized ones), which facilitates DNA signature development for detection of newly emerging pathogens. We are using suppression subtractive hybridization (SSH) as a tool to define large DNA regions specific to multiple biothreat pathogens by comparing them to genomes of the most closely related organisms. This approach has become increasingly accurate as we continue to find new, distinctive strains and ever-closer near-neighbors. With the huge costs incurred by whole genome sequencing, it is not possible to sequence each new bacterial genome. However, it is completely practical to identify genome differences in the laboratory using SSH, and becomes especially useful when comparing new strains to previously sequenced genomes.

  19. File list: Oth.Unc.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.20.DNA-RNA_hybrids.AllCell.bed ...

  20. File list: Oth.YSt.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.20.DNA-RNA_hybrids.AllCell.bed ...

  1. File list: Oth.Unc.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.50.DNA-RNA_hybrids.AllCell.bed ...

  2. File list: Oth.YSt.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.10.DNA-RNA_hybrids.AllCell.bed ...

  3. File list: Oth.Unc.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.10.DNA-RNA_hybrids.AllCell.bed ...

  4. File list: Oth.Unc.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.05.DNA-RNA_hybrids.AllCell.bed ...

  5. File list: Oth.ALL.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.DNA-RNA_hybrids.AllCell.bed ...

  6. File list: Oth.ALL.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.DNA-RNA_hybrids.AllCell.bed ...

  7. File list: Oth.YSt.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.05.DNA-RNA_hybrids.AllCell.bed ...

  8. File list: Oth.ALL.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.DNA-RNA_hybrids.AllCell.bed ...

  9. File list: Oth.ALL.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.DNA-RNA_hybrids.AllCell.bed ...

  10. File list: Oth.YSt.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.50.DNA-RNA_hybrids.AllCell.bed ...

  11. Multilayer DNA Origami Packed on Hexagonal and Hybrid Lattices

    Science.gov (United States)

    Ke, Yonggang; Voigt, Niels V.; Gothelf, Kurt V.; Shih, William M.

    2012-01-01

    “Scaffolded DNA origami” has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry. Here we report successful folding of multilayer DNA origami with helices arranged on a close-packed hexagonal lattice. This arrangement yields a higher density of helical packing and therefore higher resolution of spatial addressing than has been shown previously. We also demonstrate hybrid multilayer DNA origami with honeycomb-lattice, square-lattice, and hexagonal lattice packing of helices all in one design. The availability of hexagonal close packing of helices extends our ability to build complex structures using DNA nanotechnology. PMID:22187940

  12. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    OpenAIRE

    Choi, Yeonim; Wang, Hye-young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae; Lee, Hyeyoung

    2013-01-01

    Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specifi...

  13. Fluorescence in situ hybridization for the tissue detection of bacterial pathogens associated with porcine infections

    DEFF Research Database (Denmark)

    Jensen, Henrik Elvang; Jensen, Louise Kruse; Barington, Kristiane

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  14. Fluorescence In Situ Hybridization for the Tissue Detection of Bacterial Pathogens Associated with Porcine Infections

    DEFF Research Database (Denmark)

    Elvang Jensen, Henrik; Jensen, Louise Kruse; Barington, Kristiane

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  15. Microwave-induced inactivation of DNA-based hybrid catalyst in asymmetric catalysis.

    Science.gov (United States)

    Zhao, Hua; Shen, Kai

    2016-03-01

    DNA-based hybrid catalysts have gained strong interests in asymmetric reactions. However, to maintain the high enantioselectivity, these reactions are usually conducted at relatively low temperatures (e.g. DNA-based hybrid catalyst even at low temperatures (such as 5 °C). Circular dichroism (CD) spectra and gel electrophoresis of DNA suggest that microwave exposure degrades DNA molecules and disrupts DNA double-stranded structures, causing changes of DNA-metal ligand binding properties and thus poor DNA catalytic performance.

  16. DNA hybridization sensing for cytogenetic analysis

    DEFF Research Database (Denmark)

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

    2013-01-01

    Cytogenetic analysis focuses on studying the cell structure, mainly in respect to chromosome content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders, but are also associated with heametological malignancies. Chromosome translocations...... for cheaper detection a label-free approach has been investigated using electrochemical impedance spectroscopy as a sensing method. We present here our recent results in regards to DNA sensing on metallic and conductive polymer electrodes for translocation detection. Our sensors are inexpensive and can...... be successfully applied in cytogenetic analysis as a replacement of standard techniques....

  17. 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.

  18. The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis

    Science.gov (United States)

    Pryszcz, Leszek P.; Németh, Tibor; Saus, Ester; Ksiezopolska, Ewa; Hegedűsová, Eva; Nosek, Jozef; Wolfe, Kenneth H.; Gacser, Attila; Gabaldón, Toni

    2015-01-01

    Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids. PMID:26517373

  19. The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis.

    Directory of Open Access Journals (Sweden)

    Leszek P Pryszcz

    2015-10-01

    Full Text Available Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids.

  20. Shape changing thin films powered by DNA hybridization

    Science.gov (United States)

    Shim, Tae Soup; Estephan, Zaki G.; Qian, Zhaoxia; Prosser, Jacob H.; Lee, Su Yeon; Chenoweth, David M.; Lee, Daeyeon; Park, So-Jung; Crocker, John C.

    2017-01-01

    Active materials that respond to physical and chemical stimuli can be used to build dynamic micromachines that lie at the interface between biological systems and engineered devices. In principle, the specific hybridization of DNA can be used to form a library of independent, chemically driven actuators for use in such microrobotic applications and could lead to device capabilities that are not possible with polymer- or metal-layer-based approaches. Here, we report shape changing films that are powered by DNA strand exchange reactions with two different domains that can respond to distinct chemical signals. The films are formed from DNA-grafted gold nanoparticles using a layer-by-layer deposition process. Films consisting of an active and a passive layer show rapid, reversible curling in response to stimulus DNA strands added to solution. Films consisting of two independently addressable active layers display a complex suite of repeatable transformations, involving eight mechanochemical states and incorporating self-righting behaviour.

  1. Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample.

    Science.gov (United States)

    Khemthongcharoen, Numfon; Wonglumsom, Wijit; Suppat, Assawapong; Jaruwongrungsee, Kata; Tuantranont, Adisorn; Promptmas, Chamras

    2015-01-15

    Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called "Cholera". Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250 µm long and 50 µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5' biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50 µg/mL and 1 µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25 pg or 14 nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835 cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

    DEFF Research Database (Denmark)

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

    2011-01-01

    We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization...... modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having...... to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple...

  3. Photoinduced Reductive Electron Transfer in LNA:DNA Hybrids

    DEFF Research Database (Denmark)

    Wenge, Ulrike; Wengel, Jesper; Wagenknecht, Hans-Achim

    2012-01-01

    Lock it, but not too much: LNA units (locked or bridging nucleic acids) in LNA:DNA hybrids lead to a negative effect on electron transfer (ET), but they also force the nucleic acid structure in the A-type double helix, which allows a better base stacking than the normal B-type and thus positively...... influences the ET. This result is significant for the design of nucleic acids of molecular electronics....

  4. 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.

  5. Direct detection of expanded trinucleotide repeats using DNA hybridization techniques

    Energy Technology Data Exchange (ETDEWEB)

    Petronis, A.; Tatuch, Y.; Kennedy, J.L. [Univ. of Toronto (Canada)] [and others

    1994-09-01

    Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in several neuropsychiatric diseases, and they may play a similar role in other disorders. To our knowledge, a method that detects expanded trinucleotide sequences with the opportunity for direct localization and cloning has not been achieved. We have developed a set of hybridization-based methods for direct detection of unstable DNA expansion. Our analysis of myotonic dystrophy patients that possess different degrees of (CTG){sub n} expansion, versus unaffected controls, has demonstrated the identification of the trinucleotide instability site without any prior information regarding genetic map location. High stringency modified Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed us to detect the DNA fragment containing the expansion in myotonic dystrophy patients. The same probe was used for fluorescent in situ hybridization and several regions of (CTG){sub n}/(CAG){sub n} repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. These strategies can be applied to directly clone genes involved in disorders caused by unstable DNA.

  6. Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species.

    Science.gov (United States)

    Menardo, Fabrizio; Praz, Coraline R; Wyder, Stefan; Ben-David, Roi; Bourras, Salim; Matsumae, Hiromi; McNally, Kaitlin E; Parlange, Francis; Riba, Andrea; Roffler, Stefan; Schaefer, Luisa K; Shimizu, Kentaro K; Valenti, Luca; Zbinden, Helen; Wicker, Thomas; Keller, Beat

    2016-02-01

    Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture.

  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. Flow cytometry-based DNA hybridization and polymorphism analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.

    1998-07-01

    Functional analysis of the humane genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well-suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. The authors are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. The approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advances of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  10. Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

    Science.gov (United States)

    Ng, L K; Kingombe, C I; Yan, W; Taylor, D E; Hiratsuka, K; Malik, N; Garcia, M M

    1997-11-01

    Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that

  11. Label-free monitoring of individual DNA hybridization using SERS

    Science.gov (United States)

    Qi, Ji; Zeng, Jianbo; Zhao, Fusheng; Santos, Greggy M.; Lin, Steven Hsesheng; Raja, Balakrishnan; Strych, Ulrich; Willson, Richard C.; Shih, Wei-Chuan

    2015-03-01

    Sequence-specific detection of DNA hybridization at the single-molecule level has been instrumental and gradually become a ubiquitous tool in a wide variety of biological and biomedical applications such as clinical diagnostics, biosensors, and drug development. Label-free and amplification-free schemes are of particular interest because they could potentially provide in situ monitoring of individual hybridization events, which may lead to techniques for discriminating subtle variations due to single-base modification without stringency control or repetitive thermal cycling. Surface-enhanced Raman spectroscopy (SERS) has been widely used for molecular detection and identification by exploiting the localized surface plasmon resonance effect when the target molecules are near gold or silver nanostructures. However, effective and robust SERS assays have yet become a reality for trace detection. Recently, we have developed a SERS substrate by shaping nanoporous gold thin films into monolithic submicron disks, called nanoporous gold disks (NPGD). Here we demonstrate in situ monitoring of the same immobilized ssDNA molecules and their individual hybridization events.

  12. Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

    DEFF Research Database (Denmark)

    Eklund, Aron Charles; Friis, Pia; Wernersson, Rasmus;

    2010-01-01

    DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve...

  13. Computational analyses of ancient pathogen DNA from herbarium samples: challenges and prospects

    Science.gov (United States)

    Yoshida, Kentaro; Sasaki, Eriko; Kamoun, Sophien

    2015-01-01

    The application of DNA sequencing technology to the study of ancient DNA has enabled the reconstruction of past epidemics from genomes of historically important plant-associated microbes. Recently, the genome sequences of the potato late blight pathogen Phytophthora infestans were analyzed from 19th century herbarium specimens. These herbarium samples originated from infected potatoes collected during and after the Irish potato famine. Herbaria have therefore great potential to help elucidate past epidemics of crops, date the emergence of pathogens, and inform about past pathogen population dynamics. DNA preservation in herbarium samples was unexpectedly good, raising the possibility of a whole new research area in plant and microbial genomics. However, the recovered DNA can be extremely fragmented resulting in specific challenges in reconstructing genome sequences. Here we review some of the challenges in computational analyses of ancient DNA from herbarium samples. We also applied the recently developed linkage method to haplotype reconstruction of diploid or polyploid genomes from fragmented ancient DNA. PMID:26442080

  14. Glycidol-carbohydrate hybrids: a new family of DNA alkylating agents.

    Science.gov (United States)

    Toshima, Kazunobu; Okuno, Yukiko; Matsumura, Shuichi

    2003-10-06

    Novel and chiral glycidol-carbohydrate hybrids possessing an epoxy group as a DNA alkylating moiety were designed and synthesized. These artificial hybrids selectively alkylated DNA at the N-7 sites of the guanines and cleaved DNA without any additives. The binding ability of the glycidol was significantly enhanced by the attachment of the carbohydrate.

  15. 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...

  16. Free energy estimation of short DNA duplex hybridizations

    Directory of Open Access Journals (Sweden)

    Leger Serge

    2010-02-01

    Full Text Available Abstract Background Estimation of DNA duplex hybridization free energy is widely used for predicting cross-hybridizations in DNA computing and microarray experiments. A number of software programs based on different methods and parametrizations are available for the theoretical estimation of duplex free energies. However, significant differences in free energy values are sometimes observed among estimations obtained with various methods, thus being difficult to decide what value is the accurate one. Results We present in this study a quantitative comparison of the similarities and differences among four published DNA/DNA duplex free energy calculation methods and an extended Nearest-Neighbour Model for perfect matches based on triplet interactions. The comparison was performed on a benchmark data set with 695 pairs of short oligos that we collected and manually curated from 29 publications. Sequence lengths range from 4 to 30 nucleotides and span a large GC-content percentage range. For perfect matches, we propose an extension of the Nearest-Neighbour Model that matches or exceeds the performance of the existing ones, both in terms of correlations and root mean squared errors. The proposed model was trained on experimental data with temperature, sodium and sequence concentration characteristics that span a wide range of values, thus conferring the model a higher power of generalization when used for free energy estimations of DNA duplexes under non-standard experimental conditions. Conclusions Based on our preliminary results, we conclude that no statistically significant differences exist among free energy approximations obtained with 4 publicly available and widely used programs, when benchmarked against a collection of 695 pairs of short oligos collected and curated by the authors of this work based on 29 publications. The extended Nearest-Neighbour Model based on triplet interactions presented in this work is capable of performing accurate

  17. Telomeres in ICF syndrome cells are vulnerable to DNA damage due to elevated DNA:RNA hybrids

    Science.gov (United States)

    Sagie, Shira; Toubiana, Shir; Hartono, Stella R.; Katzir, Hagar; Tzur-Gilat, Aya; Havazelet, Shany; Francastel, Claire; Velasco, Guillaume; Chédin, Frédéric; Selig, Sara

    2017-01-01

    DNA:RNA hybrids, nucleic acid structures with diverse physiological functions, can disrupt genome integrity when dysregulated. Human telomeres were shown to form hybrids with the lncRNA TERRA, yet the formation and distribution of these hybrids among telomeres, their regulation and their cellular effects remain elusive. Here we predict and confirm in several human cell types that DNA:RNA hybrids form at many subtelomeric and telomeric regions. We demonstrate that ICF syndrome cells, which exhibit short telomeres and elevated TERRA levels, are enriched for hybrids at telomeric regions throughout the cell cycle. Telomeric hybrids are associated with high levels of DNA damage at chromosome ends in ICF cells, which are significantly reduced with overexpression of RNase H1. Our findings suggest that abnormally high TERRA levels in ICF syndrome lead to accumulation of telomeric hybrids that, in turn, can result in telomeric dysfunction. PMID:28117327

  18. Direct Electrical Detection of DNA Hybridization Based on Electrolyte-Gated Graphene Field-Effect Transistor

    Science.gov (United States)

    Ohno, Yasuhide; Okamoto, Shogo; Maehashi, Kenzo; Matsumoto, Kazuhiko

    2013-11-01

    DNA hybridization was electrically detected by graphene field-effect transistors. Probe DNA was modified on the graphene channel by a pyrene-based linker material. The transfer characteristic was shifted by the negative charges on the probe DNA, and the drain current was changed by the full-complementary DNA while no current change was observed after adding noncomplementary DNA, indicating that the graphene field-effect transistor detected the DNA hybridization. In addition, the number of DNAs was estimated by the simple plate capacitor model. As a result, one probe DNA was attached on the graphene channel per 10×10 nm2, indicating their high density functionalization. We estimated that 30% of probe DNA on the graphene channel was hybridized with 200 nM full-complementary DNA while only 5% of probe DNA was bound to the noncomplementary DNA. These results will help to pave the way for future biosensing applications based on graphene FETs.

  19. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    Science.gov (United States)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  20. Molecular Rigidity and Entropy-Enthalpy Compensation in DNA Hybridization

    Science.gov (United States)

    Douglas, Jack; Vargas-Lara, Fernando

    2015-03-01

    Entropy-enthalpy compensation (EEC) is a general and relatively poorly understood pattern in the energetic parameters governing both binding constants and relaxation processes in condensed matter. After defining the basic phenomenology, we focus on how polymer additives, chain confinement, chain length variation affect a well-studied molecular binding process, the hybridization of duplex DNA. Our study is based on a coarse-grained model of DNA that does treat water explicitly. We find that both crowding due to polymer additives and geometrical confinement lead to a change of the effective chain rigidity and that changes in DNA generally lead to a pattern entropy-enthalpy compensation in the DNA association similar to experimental observations. Modulation of the rigidity of binding specifies by constraints associated with chain structure or environmental conditions can greatly influence both the location and cooperativity of molecular binding transition and the relative enthalpy and entropy contributions to the free energy of binding. Entropy-enthalpy compensation arises in numerous synthetic and biological molecular binding processes and we suggest that that changes in molecular rigidity might provide a common explanation of this ubiquitous phenomenon.

  1. Kinetics and thermodynamics of DNA, RNA, and hybrid duplex formation.

    Science.gov (United States)

    Rauzan, Brittany; McMichael, Elizabeth; Cave, Rachel; Sevcik, Lesley R; Ostrosky, Kara; Whitman, Elisabeth; Stegemann, Rachel; Sinclair, Audra L; Serra, Martin J; Deckert, Alice A

    2013-02-05

    The rates of duplex formation for two octamers of DNA (5' d-CACGGCTC/5' d-GAGCCGTG and 5' d-CACAGCAC/5' d-GTGCTGTG), the homologous RNA, and both sets of hybrids in 1 M NaCl buffer have been measured using stopped-flow spectroscopy. In addition, the thermodynamic parameters, ΔH° and ΔS°, have been determined for the same sequences under the same buffer conditions using optical melting techniques. These data reveal a linear free energy relationship between the free energy of activation for denaturation and the change in free energy for formation of the duplexes. This relationship indicates that these duplex formation reactions occur through a common unstructured transition state that is more similar to the single strands in solution than to the ensuing duplex. In addition, these data confirm that the greater stability of RNA duplexes relative to that of homologous DNA and hybrid duplexes is controlled by the denaturation rate and not the duplex formation rate.

  2. Methods for assessing DNA hybridization of PNA-TiO2 nanoconjugates

    Science.gov (United States)

    Brown, Eric M. B.; Paunesku, Tatjana; Wu, AiGuo; Thurn, K. Ted; Haley, Benjamin; Clark, Jimmy; Priester, Taisa; Woloschak, Gayle E.

    2008-01-01

    We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO2) nanoconjugates and the several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes, which hybridize to single strand DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double strand DNA (dsDNA), and form different PNA-DNA complexes. Previously, we developed a DNA-TiO2 nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner, with the ability to cleave DNA when excited by electromagnetic radiation, but susceptible to degradation which may lower its intracellular targeting efficiency and retention time. PNA-TiO2 nanoconjugates described herein hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid and inexpensive, sequence-specific precipitation of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO2 nanoconjugates are enhanced which provides essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO2-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems. PMID:18786502

  3. Pathogenic and Epiphenomenal Anti-DNA Antibodies in SLE

    Directory of Open Access Journals (Sweden)

    Mirjana Pavlovic

    2010-01-01

    Full Text Available The discoveries of natural and the development of manufactured highly efficient catalytic antibodies (abzymes opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy and prevention (vaccination, of cancer, AIDS, autoimmune diseases. A special entity of naturally occurring DNA hydrolytic anti-DNA antibodies is emerging within past decades linked to autoimmune and lymphoproliferative disorders, such as systemic lupus erythematosus (SLE, multiple sclerosis (MS, Sjogren Syndrome (SS, B - Chronic lymphocytic leucosis (B-CLL, and Multiple Myeloma (MM. The origin of the antibodies is unknown. The underlying mechanisms of these activities are suggested to be penetration into the living cells and translocation in the nucleus, with recognition of the specific binding sites at particular (ss or ds DNA. There are controversies in the literature whether hydrolysis is a sequence-specific event. The interplay between anti-DNA antibodies and DNA is not yet elucidated. This molecular “twist” also suggests that anti-DNA antibodies with DNA hydrolytic capacity could be the organism's immune response to a microbial attack, with microbial DNA, or specific genes within microbial DNA sequence, as a target for neutralization. The catalytic antibody-based approach can become a key tool in selective chemotherapeutic strategies.

  4. Biophysical and electrochemical properties of Self-assembled noncovalent SWNT/DNA hybrid and electroactive nanostructure

    Science.gov (United States)

    Mirzapoor, Aboulfazl; Ranjbar, Bijan

    2017-09-01

    DNA self-assembled hybrid nanostructures are widely used in recent research in nanobiotechnology. Combination of DNA with carbon based nanoparticles such as single-walled carbon nanotube (SWNT), multi-walled carbon nanotube (MWNT) and carbon quantum dot were applied in important biological applications. Many examples of biosensors, nanowires and nanoelectronic devices, nanomachine and drug delivery systems are fabricated by these hybrid nanostructures. In this study, a new hybrid nanostructure has been fabricated by noncovalent interactions between single or double stranded DNA and SWNT nanoparticles and biophysical properties of these structures were studied comparatively. Biophysical properties of hybrid nanostructures studied by circular dichroism, UV-vis and fluorescence spectroscopy techniques. Also, electrochemical properties studied by cyclic voltammetry, linear sweep voltammetry, square wave voltammetry, choronoamperometry and impedance spectroscopy (EIS). Results revealed that the biophysical and electrochemical properties of SWNT/DNA hybrid nanostructures were different compare to ss-DNA, ds-DNA and SWNT singly. Circular dichroism results showed that ss-DNA wrapped around the nanotubes through π-π stacking interactions. The results indicated that after adding SWNT to ss-DNA and ds-DNA intensity of CD and UV-vis spectrum peaks were decreased. Electrochemical experiments indicated that the modification of single-walled carbon nanotubes by ss-DNA improves the electron transfer rate of hybrid nanostructures. It was demonstrated SWNT/DNA hybrid nanostructures should be a good electroactive nanostructure that can be used for electrochemical detection or sensing.

  5. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  6. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  7. Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria.

    OpenAIRE

    Beatty, J T; Cohen, S N

    1983-01-01

    The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or b...

  8. Conformational Selection and Induced Fit for RNA Polymerase and RNA/DNA Hybrid Backtracked Recognition

    Directory of Open Access Journals (Sweden)

    Haifeng eChen

    2015-11-01

    Full Text Available RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15 and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS P test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein.

  9. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

    Science.gov (United States)

    Lohman, Gregory J S; Zhang, Yinhua; Zhelkovsky, Alexander M; Cantor, Eric J; Evans, Thomas C

    2014-02-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

  10. rDNA-ITS sequence analysis of pathogens of cucumber downy mildew and cucumber powdery mildew

    Institute of Scientific and Technical Information of China (English)

    Na WANG; Yajun MA; Cuiyun YANG; Guanghui DAI; Zhezhi WANG

    2008-01-01

    To determine the pathogens of cucumber downy mildew and cucumber powdery mildew by molecular marker,we amplified and sequenced the rDNA-ITS region of the pathogens of cucumber downy mildew and cucumber powdery mildew collected from the Shanghai region.The intra-/interspecific sequence difference was analyzed by rDNA-ITS sequence.The results show that the length of rDNA-ITS1 and rDNA-ITS2 of cucumber downy mildew's pathogen was 141 bp and 406 bp,respectively,with GC contents of 41.13% in ITS1 and 46.8% (Minhang and Jinshan District,sml and sm2) or 46.55% (Pudong District,sm3) in ITS2.The rDNA-ITS sequence was intraspecific conservation.The interspecific difference was related with their kin relationship.The pathogen of cucumber downy mildew was identified as Pseudoperonospora cubensis by molecular marker.The length of rDNA-ITS1 and rDNA-ITS2 of cucumber powdery mildew's pathogen was 136 bp and 89 bp,respectively,with GC contents being 59.56% and 66.29%,and rDNA-ITS sequence being highly conservative in this study that was the same as Sphaerotheca cucurbitae.But the sequence difference between the strains in the Shanghai region in this study with S.fuliginea was 4.5%,which was identified by morphology.It is suggested that the pathogen of cucumber powdery mildew should be further clarified and determined.

  11. DNA extraction protocol for rapid PCR detection of pathogenic bacteria

    Science.gov (United States)

    Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organ...

  12. Detection of human papillomavirus DNA by the hybrid capture assay

    Directory of Open Access Journals (Sweden)

    Carvalho Maria Odete O.

    2003-01-01

    Full Text Available Human Papillomavirus (HPV infection is the main cause of cervical cancers and cervical intraepithelial neoplasias (CIN worldwide. Consequently, it would be useful to evaluate HPV testing to screen for cervical cancer. Recently developed, the second-generation Hybrid Capture (HCA II test is a non-radioactive, relatively rapid, liquid hybridization assay designed to detect 18 HPV types, divided into high and low-risk groups. We evaluated 1055 women for HPV infection with the HCA II test. Five hundred and ten (48.3% of these women had HPV infection; 60 (11.8% had low cancer-risk HPV DNA; 269 (52.7% had high-risk HPV types and 181 (35.5% had both groups. Hence, 450 women (88.2% in this HPV-infected group had at least one high risk HPV type, and were therefore considered to be at high risk for cancer. Among the group with Papanicolaou (Pap test results, the overall prevalence of HPV DNA was 58.4%. Significant differences in HPV infection of the cervix were detected between Pap I (normal smears and Pap IV (carcinomas (p<0.0001. Values of HPV viral load obtained for Pap I and SILs were significantly different, with an upward trend (p<0.0001, suggesting a positive correlation between high viral load values and risk of SIL. Because of the high costs of the HCA II test, its use for routine cervical mass screening cannot be recommended in poor countries. Nevertheless, it is a useful tool when combined with cytology, diagnosing high-risk infections in apparently normal tissues. Use of this technique could help reduce the risk of cancer.

  13. DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases.

    Science.gov (United States)

    Cao, Boyang; Wang, Suwei; Tian, Zhenyang; Hu, Pinliang; Feng, Lu; Wang, Lei

    2015-01-01

    This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.

  14. 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.

  15. Karyotyping of Brassica oleracea L.based on rDNA and Cot-1 DNA fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    WANG Taixia; WU Chunhong; HUANG Jinyong; WEI Wenhui

    2007-01-01

    To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.

  16. Hole Transport in A-form DNA/RNA Hybrid Duplexes

    Science.gov (United States)

    Wong, Jiun Ru; Shao, Fangwei

    2017-01-01

    DNA/RNA hybrid duplexes are prevalent in many cellular functions and are an attractive target form for electrochemical biosensing and electric nanodevice. However the electronic conductivities of DNA/RNA hybrid duplex remain relatively unexplored and limited further technological applications. Here cyclopropyl-modified deoxyribose- and ribose-adenosines were developed to explore hole transport (HT) in both DNA duplex and DNA/RNA hybrids by probing the transient hole occupancies on adenine tracts. HT yields through both B-form and A-form double helixes displayed similar shallow distance dependence, although the HT yields of DNA/RNA hybrid duplexes were lower than those of DNA duplexes. The lack of oscillatory periods and direction dependence in HT through both helixes implied efficient hole propagation can be achieved via the hole delocalization and coherent HT over adenine tracts, regardless of the structural variations.

  17. Selective Inactivation of Resistant Gram-Positive Pathogens with a Light-Driven Hybrid Nanomaterial.

    Science.gov (United States)

    Grüner, Malte; Tuchscherr, Lorena; Löffler, Bettina; Gonnissen, Dominik; Riehemann, Kristina; Staniford, Mark C; Kynast, Ulrich; Strassert, Cristian A

    2015-09-23

    Herein, we present a straightforward strategy to disperse highly insoluble photosensitizers in aqueous environments, without major synthetic efforts and keeping their photosensitizing abilities unaffected. A layered nanoclay was employed to adsorb and to solubilize a highly efficient yet hydrophobic Si(IV) phthalocyaninate in water. The aggregation of the photoactive dye was correlated with its photophysical properties, particularly with the ability to produce highly cytotoxic singlet oxygen. Moreover, the resulting hybrid nanomaterial is able to selectively photoinactivate Gram-positive pathogens, due to local interactions between the bacterial membranes and the negatively charged nanodiscs. Nanotoxicity assays confirmed its innocuousness toward eukaryotic cells, showing that it constitutes a new class of "phototriggered magic bullet" for the inactivation of pathogens in phototherapy, as well as in the development of coatings for self-disinfecting surfaces.

  18. Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae.

    Science.gov (United States)

    Schwenkbier, Lydia; Pollok, Sibyll; Rudloff, Anne; Sailer, Sebastian; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-10-07

    A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.

  19. Pathogenic microbial ancient DNA: a problem or an opportunity?

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2006-01-01

    such as independent replication and cloning. Only 3 out of 47 studies have performed independent replication at all (Drancourt & Raoult 2005), and none of the studies (Spigelman et al. 2002; Fletcher et al. 2003a,b; Donoghue et al. 2005) mentioned by Donoghue & Spigelman (2005) as following aDNA criteria included...

  20. 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.

  1. File list: Oth.NoD.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.05.DNA-RNA_hybrids.AllCell.bed ...

  2. File list: Oth.NoD.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.10.DNA-RNA_hybrids.AllCell.bed ...

  3. File list: Oth.NoD.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.50.DNA-RNA_hybrids.AllCell.bed ...

  4. File list: Oth.NoD.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.20.DNA-RNA_hybrids.AllCell.bed ...

  5. Nuclear and mitochondrial DNA analysis reveals that hybridization between Fasciola hepatica and Fasciola gigantica occurred in China.

    Science.gov (United States)

    Ichikawa-Seki, Madoka; Peng, Mao; Hayashi, Kei; Shoriki, Takuya; Mohanta, Uday Kumar; Shibahara, Toshiyuki; Itagaki, Tadashi

    2017-02-01

    The well-known pathogens of fasciolosis, Fasciola hepatica (Fh) and Fasciola Gigantica (Fg), possess abundant mature sperms in their seminal vesicles, and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The aim of this study was to reveal the origin of aspermic Fasciola flukes. The nuclear single copy markers, phosphoenolpyruvate carboxykinase and DNA polymerase delta, were employed for analysis of Fasciola species from China. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the Fh/Fg type, which includes DNA fragments of both F. hepatica and F. gigantica. China can be regarded as the cradle of the interspecific hybridization because F. hepatica and F. gigantica were detected in the northern and southern parts of China, respectively, and hybrids flukes were distributed between the habitats of the two species. The Chinese origin was supported by the fact that a larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes was detected in Chinese aspermic Fasciola populations than in aspermic populations from the neighbouring countries. Hereafter, 'aspermic' Fasciola flukes should be termed as 'hybrid' Fasciola flukes.

  6. [Determination of the structural integrity of DNA from pathogenic enterobacteria].

    Science.gov (United States)

    Tets, V V; Ivanov, S D

    1985-06-01

    Among the enterobacterial strains under study, more organisms in the stationary phase of growth have been found to have nicks in their DNA than those in the exponential phase. Bacteria less sensitive to ultraviolet irradiation have the least number of nicks in each phase of growth. The number of nicks in different strains belonging to the serovar is sufficiently stable. Virulent and avirulent forms show no difference in this characteristic.

  7. Thermodynamically unfavorable DNA hybridizations can be made to occur by a water to ice phase change.

    Science.gov (United States)

    Krissanaprasit, Abhichart; Guajardo, Cristian; Somasundrum, Mithran; Surareungchai, Werasak

    2013-02-01

    In an apparent contradiction to Debye-Hückel theory, it was possible to hybridize DNA in solutions of Milli-Q water (resistivity>18MΩcm(-1)) containing no added ions. This was demonstrated by hybridizing four bi-complementary DNA sequences to form an 'X' shape, as indicated by acrylamide gel electrophoresis. The requirement for hybridization was that a water-to-ice phase change should occur. Comparative experiments, using freezing by liquid nitrogen and thawing at different temperatures, showed that hybridization could take place during either the freezing or thawing process provided either was slow enough. We speculate that the low solubility of DNA in ice creates liquid inclusions of extremely high DNA and counter-ion concentration prior to complete freezing, and that hence in these inclusions hybridization was actually in accordance with Debye-Hückel theory.

  8. Application of DNA hybridization techniques in the assessment of diarrheal disease among refugess in Thailand. [Shigella; Escherichia coli; Campylobacter; Cryptosporidium

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, D.N.; Echeverria, P.; Pitarangsi, C.; Seriwatana, J.; Sethabutr, O.; Bodhidatta, L.; Brown, C.; Herrmann, J.E.; Blacklow, N.R.

    1988-01-01

    The epidemiology and etiology of acute diarrheal disease were determined in a Hmong refugee camp on the Thai-Laotian border from April 11 to May 14, 1985. DNA hybridization techniques were used to detect Shigella species, enteroinvasive Escherichia coli, and enterotoxigenic E. coli. A monoclonal enzyme-linked immunosorbent assay was used to detect rotavirus, and standard microbiology was used to detect other enteropathogens. The age-specific diarrheal disease rates were 47 episodes per month per 1000 children less than five years old and 113 episodes per month per 1000 children less than one year old. Rotavirus, enterotoxigenic E. coli, Campylobacter, and Cryptosporidium were the predominant pathogens in children less than two years old. The DNA probe hybridized with 94% of 31 specimens identified as enterotoxigenic E. coli by the standard assays and with none of the specimens in which the standard assays were negative. The probe for Shigella and enteroinvasive E. coli hybridized in eight of 10 stools that contained Shigella and four of 314 stools from which Shigella and enteroinvasive E. coli were not isolated. The use of DNA probes allows specimens to be collected in remote areas with a minimum amount of equipment and technical expertise so that they can be easily transported to a central laboratory for further processing.

  9. The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. infestans.

    Science.gov (United States)

    Goss, Erica M; Cardenas, Martha E; Myers, Kevin; Forbes, Gregory A; Fry, William E; Restrepo, Silvia; Grünwald, Niklaus J

    2011-01-01

    Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.

  10. The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. infestans.

    Directory of Open Access Journals (Sweden)

    Erica M Goss

    Full Text Available Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.

  11. Genetic Evidence for Elevated Pathogenicity of Mitochondrial DNA Heteroplasmy in Autism Spectrum Disorder.

    Directory of Open Access Journals (Sweden)

    Yiqin Wang

    2016-10-01

    Full Text Available Increasing clinical and biochemical evidence implicate mitochondrial dysfunction in the pathophysiology of Autism Spectrum Disorder (ASD, but little is known about the biological basis for this connection. A possible cause of ASD is the genetic variation in the mitochondrial DNA (mtDNA sequence, which has yet to be thoroughly investigated in large genomic studies of ASD. Here we evaluated mtDNA variation, including the mixture of different mtDNA molecules in the same individual (i.e., heteroplasmy, using whole-exome sequencing data from mother-proband-sibling trios from simplex families (n = 903 where only one child is affected by ASD. We found that heteroplasmic mutations in autistic probands were enriched at non-polymorphic mtDNA sites (P = 0.0015, which were more likely to confer deleterious effects than heteroplasmies at polymorphic mtDNA sites. Accordingly, we observed a ~1.5-fold enrichment of nonsynonymous mutations (P = 0.0028 as well as a ~2.2-fold enrichment of predicted pathogenic mutations (P = 0.0016 in autistic probands compared to their non-autistic siblings. Both nonsynonymous and predicted pathogenic mutations private to probands conferred increased risk of ASD (Odds Ratio, OR[95% CI] = 1.87[1.14-3.11] and 2.55[1.26-5.51], respectively, and their influence on ASD was most pronounced in families with probands showing diminished IQ and/or impaired social behavior compared to their non-autistic siblings. We also showed that the genetic transmission pattern of mtDNA heteroplasmies with high pathogenic potential differed between mother-autistic proband pairs and mother-sibling pairs, implicating developmental and possibly in utero contributions. Taken together, our genetic findings substantiate pathogenic mtDNA mutations as a potential cause for ASD and synergize with recent work calling attention to their unique metabolic phenotypes for diagnosis and treatment of children with ASD.

  12. Genetic Evidence for Elevated Pathogenicity of Mitochondrial DNA Heteroplasmy in Autism Spectrum Disorder

    Science.gov (United States)

    Wang, Yiqin; Picard, Martin; Gu, Zhenglong

    2016-01-01

    Increasing clinical and biochemical evidence implicate mitochondrial dysfunction in the pathophysiology of Autism Spectrum Disorder (ASD), but little is known about the biological basis for this connection. A possible cause of ASD is the genetic variation in the mitochondrial DNA (mtDNA) sequence, which has yet to be thoroughly investigated in large genomic studies of ASD. Here we evaluated mtDNA variation, including the mixture of different mtDNA molecules in the same individual (i.e., heteroplasmy), using whole-exome sequencing data from mother-proband-sibling trios from simplex families (n = 903) where only one child is affected by ASD. We found that heteroplasmic mutations in autistic probands were enriched at non-polymorphic mtDNA sites (P = 0.0015), which were more likely to confer deleterious effects than heteroplasmies at polymorphic mtDNA sites. Accordingly, we observed a ~1.5-fold enrichment of nonsynonymous mutations (P = 0.0028) as well as a ~2.2-fold enrichment of predicted pathogenic mutations (P = 0.0016) in autistic probands compared to their non-autistic siblings. Both nonsynonymous and predicted pathogenic mutations private to probands conferred increased risk of ASD (Odds Ratio, OR[95% CI] = 1.87[1.14–3.11] and 2.55[1.26–5.51], respectively), and their influence on ASD was most pronounced in families with probands showing diminished IQ and/or impaired social behavior compared to their non-autistic siblings. We also showed that the genetic transmission pattern of mtDNA heteroplasmies with high pathogenic potential differed between mother-autistic proband pairs and mother-sibling pairs, implicating developmental and possibly in utero contributions. Taken together, our genetic findings substantiate pathogenic mtDNA mutations as a potential cause for ASD and synergize with recent work calling attention to their unique metabolic phenotypes for diagnosis and treatment of children with ASD. PMID:27792786

  13. DNA topoisomerases from pathogenic fungi: targets for the discovery of antifungal drugs.

    OpenAIRE

    Shen, L L; Baranowski, J; Fostel, J.; Montgomery, D A; Lartey, P A

    1992-01-01

    DNA topoisomerases, a class of enzymes that change the topological structure of DNA, have been shown to be the target of many therapeutic agents, including antibacterial agents (quinolones) and anticancer agents. These drugs inhibit the enzyme in a unique way so that the enzyme is converted into a cellular poison. Candida albicans and Aspergillus niger are two major opportunistic fungal pathogens. Our results show that these fungi have high levels of both type I and type II topoisomerases (wi...

  14. DNA-Accelerated Catalysis of Carbene-Transfer Reactions by a DNA/Cationic Iron Porphyrin Hybrid

    NARCIS (Netherlands)

    Rioz-Martínez, Ana; Oelerich, Jens; Ségaud, Nathalie; Roelfes, Gerard

    2016-01-01

    A novel DNA-based hybrid catalyst comprised of salmon testes DNA and an iron(III) complex of a cationic meso-tetrakis(N-alkylpyridyl)porphyrin was developed. When the N-methyl substituents were placed at the ortho position with respect to the porphyrin ring, high reactivity in catalytic

  15. Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction.

    Science.gov (United States)

    Xiang, Binbin; He, Kaiyu; Zhu, Rong; Liu, Zhuoliang; Zeng, Shu; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

    2016-09-07

    DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.

  16. Computational analyses of ancient pathogen DNA from herbarium samples: challenges and prospects

    Directory of Open Access Journals (Sweden)

    Kentaro eYoshida

    2015-09-01

    Full Text Available The application of DNA sequencing technology to the study of ancient DNA has enabled the reconstruction of past epidemics from genomes of historically important plant-associated microbes. Recently, the genome sequences of the potato late blight pathogen Phytophthora infestans were analyzed from 19th century herbarium specimens. These herbarium samples originated from infected potatoes collected during and after the Irish potato famine. Herbaria have therefore great potential to help elucidate past epidemics of crops, date the emergence of pathogens, and inform about past pathogen population dynamics. DNA preservation in herbarium samples was unexpectedly good, raising the possibility of a whole new research area in plant and microbial genomics. However, the recovered DNA can be extremely fragmented resulting in specific challenges in reconstructing genome sequences. Here we review some of the challenges in computational analyses of ancient DNA from herbarium samples. We also applied the recently developed linkage method to haplotype reconstruction of diploid or polyploid genomes from fragmented ancient DNA.

  17. Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen.

    Science.gov (United States)

    Eslami, Gilda; Khalatbari-Limaki, Sepideh; Ehrampoush, Mohammad Hasan; Gholamrezaei, Mostafa; Hajimohammadi, Bahador; Oryan, Ahmad

    2017-01-01

    One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata. The larvae were collected from the sheep's visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI), and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively. The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same. Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

  18. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  19. 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.

  20. Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier

    NARCIS (Netherlands)

    J.C. Alers (Janneke); P-J. Krijtenburg (Pieter-Jaap); K.J. Vissers (Kees); H. van Dekken (Herman)

    1999-01-01

    textabstractDecalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on

  1. DNA elimination in embryogenic development of Pennisetum glaucum x Pennisetum purpureum (Poaceae) hybrids.

    Science.gov (United States)

    Nunes, J D; Azevedo, A L S; Pereira, A V; Paula, C M P; Campos, J M S; Lédo, F J S; Santos, V B

    2013-10-22

    Interspecific hybridization between Napier grass (Pennisetum purpureum), which is widely grown in Brazil for cattle forage, and pearl millet (Pennisetum glaucum) has been used as a breeding strategy for the development of improved cultivars. However, the hybrid between these two species is sterile due to its triploid condition (2n = 3x = 21 chromosomes), which hinders its use in crop breeding programs. It is known that genomic alterations result from the hybridization process. In order to measure the loss of DNA during embryo development, we used flow cytometry to estimate the nuclear DNA content of triploid and tetraploid embryos produced by interspecific hybridization between Napier grass and pearl millet. The triploid and tetraploid hybrids had a mean DNA content of 4.99-4.87 and 5.25-4.84 pg, at 10 and 30 days after pollination, respectively. The mean reduction in DNA content was higher in the tetraploid hybrids. The flow cytometry results revealed progressive genomic instability in these triploid and tetraploid hybrids, with this instability causing significant alterations in the DNA content of the hybrids.

  2. Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis

    NARCIS (Netherlands)

    Lievens, B.; Claes, L.; Vanachter, A.C.R.C.; Cammue, B.P.A.; Thomma, B.P.H.J.

    2006-01-01

    The lack of a rapid and reliable means for routine pathogen identification has been one of the main limitations in plant disease management, and has pushed the development of culture-independent, molecular approaches. Currently, DNA array technology is the most suitable technique for high-throughput

  3. Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism.

    Science.gov (United States)

    Olivares-Fuster, Oscar; Hernández-Garrido, María; Guerri, José; Navarro, Luis

    2007-06-01

    Unlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts.

  4. Dyes as bifunctional markers of DNA hybridization on surfaces and mutation detection.

    Science.gov (United States)

    García-Mendiola, Tania; Cerro, María Ramos; López-Moreno, José María; Pariente, Félix; Lorenzo, Encarnación

    2016-10-01

    The interaction of small molecules with DNA has found diagnostic and therapeutic applications. In this work, we propose the use of two different dyes, in particular Azure A and Safranine, as bifunctional markers of on-surface DNA hybridization and potent tools for screening of specific gene mutations directly in real DNA PCR amplicons extracted from blood cells. By combining spectroscopic and electrochemical methods we demonstrate that both dyes can interact with single and double stranded DNA to a different extent, allowing reliable hybridization detection. From these data, we have also elucidated the nature of the interaction. We conclude that the binding mode is fundamentally intercalative with an electrostatic component. The dye fluorescence allows their use as nucleic acid stains for the detection of on-surfaces DNA hybridization. Its redox activity is exploited in the development of selective electrochemical DNA biosensors.

  5. Advances in Human Mitochondrial Diseases Molecular Genetic Analysis of Pathogenic mtDNA Mutations.

    Science.gov (United States)

    Davidson, E; King, M P

    1997-01-01

    The mitochondrial diseases are a heterogeneous group of disorders that have been defined by specific morphological alterations in muscle and by deficits of the mitochondrial respiratory chain. The morphological hallmarks of these diseases include ragged-red fibers (an extensive proliferation of mitochondria in muscle fibers) and abnormal paracrystalline inclusions and membrane structures in mitochondria. The identification of pathogenic mutations in mitochondrial DNA (mtDNA) has resulted in a genetic classification of mitochondrial diseases. Investigations are being conducted to understand the molecular basis for the biochemical and morphological alterations of mitochondria associated with mtDNA mutations. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:16-24).

  6. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela

    2014-03-20

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A DNA origami nanorobot controlled by nucleic acid hybridization.

    Science.gov (United States)

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  8. 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.

  9. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    Science.gov (United States)

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  10. Role for RNA: DNA hybrids in origin-independent replication priming in a eukaryotic system

    OpenAIRE

    Stuckey, Ruth; García Rodriguez, Néstor; Aguilera López, Andrés; Wellinger, Ralf Erik

    2015-01-01

    DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops)...

  11. Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

    Indian Academy of Sciences (India)

    Mízia M S Silva; Igor T Cavalcanti; M Fátima Barroso; M Goreti F Sales; Rosa Fireman Dutra

    2010-11-01

    The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.

  12. Enantioselective Catalysis by Using Short, Structurally Defined DNA Hairpins as Scaffold for Hybrid Catalysts.

    Science.gov (United States)

    Marek, Jasmin J; Singh, Raghvendra P; Heuer, Andreas; Hennecke, Ulrich

    2017-05-02

    A new type of DNA metal complex hybrid catalyst, which is based on single-stranded DNA oligonucleotides, is described. It was shown that oligonucleotides as short as 14 nucleotides that fold into hairpin structures are suitable as nucleic acid components for DNA hybrid catalysts. With these catalysts, excellent enantioinduction in asymmetric Diels-Alder reactions with selectivity values as high as 96 % enantiomeric excess (ee) can be achieved. Molecular dynamics simulations indicate that a rather flexible loop combined with a rigid stem region provides DNA scaffolds with these high selectivity values. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells.

    Science.gov (United States)

    Wang, Zheng; Yin, Hao; Lv, Lei; Feng, Yingying; Chen, Shaopeng; Liang, Junting; Huang, Yun; Jiang, Xiaohua; Jiang, Hanwei; Bukhari, Ihtisham; Wu, Lijun; Cooke, Howard J; Shi, Qinghua

    2014-01-01

    Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human-mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human-mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.

  14. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  15. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  16. 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.

  17. Labeling-free fluorescent detection of DNA hybridization through FRET from pyrene excimer to DNA intercalator SYBR green I.

    Science.gov (United States)

    Zhou, Ruyi; Xu, Chen; Dong, Jie; Wang, Guojie

    2015-03-15

    A novel labeling-free fluorescence complex probe has been developed for DNA hybridization detection based on fluorescence resonance energy transfer (FRET) mechanism from pyrene excimer of pyrene-functionalized poly [2-(N, N-dimethylamino) ethyl methacrylate] (PFP) to SYBR Green I (SG, a specific intercalator of double-stranded DNA) in a cost-effective, rapid and simple manner. The complex probe consists of the positively charged PFP, SG and negatively charged single-stranded DNA (ssDNA). Upon adding a complementary strand to the complex probe solution, double-stranded DNA (dsDNA) was formed, followed by the intercalation of SG into dsDNA. The pyrene excimer emission was overlapped with the absorption of SG very well and the electrostatic interactions between PFP and dsDNA kept them in close proximity, enabling efficient FRET from pyrene excimer to SG. The fluorescence of SG in the duplex DNA resulting from FRET can be successfully applied to detect DNA hybridization with high sensitivity for a very low detection limit of 10nM and excellent selectivity for detection of single base pair mismatch. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. DNA-inorganic hybrid nanovaccine for cancer immunotherapy

    Science.gov (United States)

    Zhu, Guizhi; Liu, Yijing; Yang, Xiangyu; Kim, Young-Hwa; Zhang, Huimin; Jia, Rui; Liao, Hsien-Shun; Jin, Albert; Lin, Jing; Aronova, Maria; Leapman, Richard; Nie, Zhihong; Niu, Gang; Chen, Xiaoyuan

    2016-03-01

    Cancer evolves to evade or compromise the surveillance of the immune system, and cancer immunotherapy aims to harness the immune system in order to inhibit cancer development. Unmethylated CpG dinucleotide-containing oligonucleotides (CpG), a class of potent adjuvants that activate the toll-like receptor 9 (TLR9) located in the endolysosome of many antigen-presenting cells (APCs), are promising for cancer immunotherapy. However, clinical application of synthetic CpG confronts many challenges such as suboptimal delivery into APCs, unfavorable pharmacokinetics caused by limited biostability and short in vivo half-life, and side effects associated with leaking of CpG into the systemic circulation. Here we present DNA-inorganic hybrid nanovaccines (hNVs) for efficient uptake into APCs, prolonged tumor retention, and potent immunostimulation and cancer immunotherapy. hNVs were self-assembled from concatemer CpG analogs and magnesium pyrophosphate (Mg2PPi). Mg2PPi renders hNVs resistant to nuclease degradation and thermal denaturation, both of which are demanding characteristics for effective vaccination and the storage and transportation of vaccines. Fluorophore-labeled hNVs were tracked to be efficiently internalized into the endolysosomes of APCs, where Mg2PPi was dissolved in an acidic environment and thus CpG analogs were exposed to hNVs. Internalized hNVs in APCs led to (1) elevated secretion of proinflammatory factors, and (2) elevated expression of co-stimulatory factors. Compared with molecular CpG, hNVs dramatically prolonged the tissue retention of CpG analogs and reduced splenomegaly, a common side effect of CpG. In a melanoma mouse model, two injections of hNVs significantly inhibited the tumor growth and outperformed the molecular CpG. These results suggest hNVs are promising for cancer immunotherapy.Cancer evolves to evade or compromise the surveillance of the immune system, and cancer immunotherapy aims to harness the immune system in order to inhibit

  19. A proposed mechanism of the influence of gold nanoparticles on DNA hybridization.

    Science.gov (United States)

    Sedighi, Abootaleb; Li, Paul C H; Pekcevik, Idah C; Gates, Byron D

    2014-07-22

    A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA-gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors.

  20. Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. and Curt.) arx.

    Science.gov (United States)

    Freeman, S.; Redman, R.S.; Grantham, G.; Rodriguez, R.J.

    1997-01-01

    A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

  1. Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.

  2. 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.

  3. Hybrid Structures for Surface-Enhanced Raman Scattering: DNA Origami/Gold Nanoparticle Dimer/Graphene.

    Science.gov (United States)

    Prinz, Julia; Matković, Aleksandar; Pešić, Jelena; Gajić, Radoš; Bald, Ilko

    2016-10-01

    A combination of three innovative materials within one hybrid structure to explore the synergistic interaction of their individual properties is presented. The unique electronic, mechanical, and thermal properties of graphene are combined with the plasmonic properties of gold nanoparticle (AuNP) dimers, which are assembled using DNA origami nanostructures. This novel hybrid structure is characterized by means of correlated atomic force microscopy and surface-enhanced Raman scattering (SERS). It is demonstrated that strong interactions between graphene and AuNPs result in superior SERS performance of the hybrid structure compared to their individual components. This is particularly evident in efficient fluorescence quenching, reduced background, and a decrease of the photobleaching rate up to one order of magnitude. The versatility of DNA origami structures to serve as interface for complex and precise arrangements of nanoparticles and other functional entities provides the basis to further exploit the potential of the here presented DNA origami-AuNP dimer-graphene hybrid structures.

  4. Remote toehold: a mechanism for flexible control of DNA hybridization kinetics.

    Science.gov (United States)

    Genot, Anthony J; Zhang, David Yu; Bath, Jonathan; Turberfield, Andrew J

    2011-02-23

    Hybridization of DNA strands can be used to build molecular devices, and control of the kinetics of DNA hybridization is a crucial element in the design and construction of functional and autonomous devices. Toehold-mediated strand displacement has proved to be a powerful mechanism that allows programmable control of DNA hybridization. So far, attempts to control hybridization kinetics have mainly focused on the length and binding strength of toehold sequences. Here we show that insertion of a spacer between the toehold and displacement domains provides additional control: modulation of the nature and length of the spacer can be used to control strand-displacement rates over at least 3 orders of magnitude. We apply this mechanism to operate displacement reactions in potentially useful kinetic regimes: the kinetic proofreading and concentration-robust regimes.

  5. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

    Science.gov (United States)

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P

    2015-01-01

    DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

  6. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

    Directory of Open Access Journals (Sweden)

    Vikash Verma

    Full Text Available DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

  7. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

    Science.gov (United States)

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

    2015-01-01

    DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

  8. The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

    Science.gov (United States)

    Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun

    2015-12-03

    The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

  9. Microfluidic Arrayed Lab-On-A-Chip for Electrochemical Capacitive Detection of DNA Hybridization Events.

    Science.gov (United States)

    Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

    2017-01-01

    A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events. A quantitative analysis of the hybridization events is carried out based on a sensing modeling that includes detailed analysis of energy storage and dissipation components. By calculating these components during hybridization events the device is able to demonstrate specific and dose response sensing characteristics. The developed microfluidic LOC for DNA hybridization detection offers a technology for real-time and label-free assessment of genetic markers outside of laboratory settings, such as at the point-of-care or in-field environmental monitoring.

  10. Magnetoresistive sensors for measurements of DNA hybridization kinetics - effect of TINA modifications

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt

    2017-01-01

    Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5' end (1 × TINA) or at both......We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current....... A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic...

  11. CpG DNA: A pathogenic factor in systemic lupus erythematosus?

    Energy Technology Data Exchange (ETDEWEB)

    Krieg, A.M. [Univ. of Iowa College of Medicine, Iowa City, IA (United States)

    1995-11-01

    Systemic lupus erythematosus (SLE) is a multifactorial disease of unknown etiology. Characteristic features of SLE include (1) polyclonal B cell activation, (2) overexpression of the immune stimulatory cytokine interleukin-6 (IL-6), (3) defective tolerance to self antigens, and (4) production of anti-DNA antibodies (Ab). Bacterial infection has been suspected as a triggering factor for lupus. Bacterial DNA differs from vertebrate DNA in the frequency and methylation of CpG dinucleotides. These CpG motifs in bacterial DNA induce a variety of immune effects, including (1) polyclonal activation of murine and human B cells, (2) IL-6 secretion, and (3) resistance to apoptosis, thereby potentially allowing the survival of autoreactive cells. These results suggest that microbial DNA could therefore be a pathogenic factor in SLE. SLE patients have elevated levels of circulating plasma DNA which is reportedly enriched in hypomethylated CpGs. Genomic DNA is also hypomethylated in SLE. The purpose of this review is to summarize the immune effects of CpG motifs and to present the evidence for their possible involvement in the pathogenesis of SLE. 77 refs.

  12. Magnetic particle-based sandwich sensor with DNA-modified carbon nanotubes as recognition elements for detection of DNA hybridization.

    Science.gov (United States)

    Hu, Po; Huang, Cheng Zhi; Li, Yuan Fang; Ling, Jian; Liu, Yu Ling; Fei, Liang Run; Xie, Jian Ping

    2008-03-01

    In this contribution, we design a visual sensor for DNA hybridization with DNA probe-modified magnetic particles (MPs) and multiwalled carbon nanotubes (MWNTs) without involving a visual recognition element such as fluorescent/chemiluminescent reagents. It was found that DNA probe-modified MWNTs, which could be dispersed in aqueous medium and have strong light scattering signals under the excitation of a light beam in the UV-vis region, could connect with DNA probe-modified MPs together in the presence of perfectly complementary target DNA and form a sandwich structure. In a magnetic field, the formed MP-MWNT species can easily be removed from the solution, resulting in a decrease of light scattering signals. Thus, a magnetic particle-based sandwich sensor could be developed to detect DNA hybridization by measuring the light scattering signals with DNA-modified MWNTs as recognition elements. Experiments showed that the DNA-modified MPs sensor could be reused at least 17 times and was stable for more than 6 months.

  13. The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments

    Science.gov (United States)

    Yang, ZhouJie; Yuan, GuoHua; Zhai, WeiLi; Yan, Jie; Chen, Hu

    2016-08-01

    Deoxyribonucleic acid (DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded (ss) strands, forming a double-stranded (ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.

  14. Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

    Science.gov (United States)

    Manera, M. G.; Spadavecchia, J.; Taurino, A.; Rella, R.

    2010-03-01

    The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV-vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA-Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA-DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T)15ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality.

  15. DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool

    Directory of Open Access Journals (Sweden)

    Jin-Young Park

    2009-11-01

    Full Text Available Recent advances in label free DNA hybridization sensors employing electrochemical impedance spectroscopy (EIS as a detection tool are reviewed. These sensors are based on the modulation of the blocking ability of an electrode modified with a probe DNA by an analyte, i.e., target DNA. The probe DNA is immobilized on a self-assembled monolayer, a conducting polymer film, or a layer of nanostructures on the electrode such that desired probe DNA would selectively hybridize with target DNA. The rate of charge transfer from the electrode thus modified to a redox indicator, e.g., [Fe(CN6]3–/4–, which is measured by EIS in the form of charge transfer resistance (Rct, is modulated by whether or not, as well as how much, the intended target DNA is selectively hybridized. Efforts made to enhance the selectivity as well as the sensitivity of DNA sensors and to reduce the EIS measurement time are briefly described along with brief future perspectives in developing DNA sensors.

  16. DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool

    Science.gov (United States)

    Park, Jin-Young; Park, Su-Moon

    2009-01-01

    Recent advances in label free DNA hybridization sensors employing electrochemical impedance spectroscopy (EIS) as a detection tool are reviewed. These sensors are based on the modulation of the blocking ability of an electrode modified with a probe DNA by an analyte, i.e., target DNA. The probe DNA is immobilized on a self-assembled monolayer, a conducting polymer film, or a layer of nanostructures on the electrode such that desired probe DNA would selectively hybridize with target DNA. The rate of charge transfer from the electrode thus modified to a redox indicator, e.g., [Fe(CN)6]3−/4−, which is measured by EIS in the form of charge transfer resistance (Rct), is modulated by whether or not, as well as how much, the intended target DNA is selectively hybridized. Efforts made to enhance the selectivity as well as the sensitivity of DNA sensors and to reduce the EIS measurement time are briefly described along with brief future perspectives in developing DNA sensors. PMID:22303136

  17. Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA-DNA hybridization.

    Science.gov (United States)

    Gnat, Sebastian; Małek, Wanda; Oleńska, Ewa; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Rogalski, Jerzy; Wójcik, Magdalena

    2016-04-01

    In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.

  18. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  19. Ultrasensitive colorimetric detection of circulating tumor DNA using hybridization chain reaction and the pivot of triplex DNA

    Science.gov (United States)

    Li, Ruimin; Zou, Li; Luo, Yanwei; Zhang, Manjun; Ling, Liansheng

    2017-03-01

    This work presents an amplified colorimetric biosensor for circulating tumor DNA (ctDNA), which associates the hybridization chain reaction (HCR) amplification with G-Quadruplex DNAzymes activity through triplex DNA formation. In the presence of ctDNA, HCR occurs. The resulting HCR products are specially recognized by one sequence to include one GGG repeat and the other containing three GGG repeats, through the synergetic effect of triplex DNA and asymmetrically split G-Quadruplex forming. Such design takes advantage of the amplification property of HCR and the high peroxidase-like catalytic activity of asymmetrically split G-Quadruplex DNAzymes by means of triplex DNA formation, which produces color signals in the presence of ctDNA. Nevertheless, in the absence of ctDNA, no HCR happens. Thus, no triplex DNA and G-Quadruplex structure is formed, producing a negligible background. The colorimetric sensing platform is successfully applied in complex biological environments such as human blood plasma for ctDNA detection, with a detection limit corresponding to 0.1 pM. This study unambiguously uses triplex DNA forming as the pivot to integrate nucleic acid amplification and DNAzymes for producing a highly sensitive signal with low background.

  20. UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis.

    Science.gov (United States)

    Kunz, Bernard A; Dando, Paige K; Grice, Desma M; Mohr, Peter G; Schenk, Peer M; Cahill, David M

    2008-10-01

    Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280-320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

  1. 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.

  2. 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.

  3. Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system.

    Science.gov (United States)

    Maturos, Thitima; Pogfay, Tawee; Rodaree, Kiattimant; Chaotheing, Sastra; Jomphoak, Apichai; Wisitsoraat, Anurat; Suwanakitti, Nattida; Wongsombat, Chayapat; Jaruwongrungsee, Kata; Shaw, Philip; Kamchonwongpaisan, Sumalee; Tuantranont, Adisorn

    2012-01-07

    Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2013-06-14

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

  5. Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum.

    Science.gov (United States)

    Lohnoo, Tassanee; Jongruja, Nujarin; Rujirawat, Thidarat; Yingyon, Wanta; Lerksuthirat, Tassanee; Nampoon, Umporn; Kumsang, Yothin; Onpaew, Pornpit; Chongtrakool, Piriyaporn; Keeratijarut, Angsana; Brandhorst, Tristan T; Krajaejun, Theerapong

    2014-03-01

    The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at the molecular level could lead to better means of infection control High quality genomic DNA (gDNA) is needed for molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies, phylogenetic analysis, and molecular genetics assays. To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for fungal gDNA preparation. Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction, rapid-extraction, and salt-extraction protocols. The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale gDNA samples. Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods, the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or liquid nitrogen.

  6. Graphene coated fiber optic surface plasmon resonance biosensor for the DNA hybridization detection: Simulation analysis

    Science.gov (United States)

    Shushama, Kamrun Nahar; Rana, Md. Masud; Inum, Reefat; Hossain, Md. Biplob

    2017-01-01

    In this paper, a graphene coated optical fiber surface plasmon resonance (SPR) biosensor is presented for the detection of DNA Hybridization. For the proposed sensor, a four layer model (fiber core /metal /sensing layer /sample) where a sheet of graphene (biomolecular recognition elements (BRE)) acting as a sensing layer is coated around the gold film because graphene enhances the sensitivity of fiber optic SPR biosensor. Numerical analysis shows the variation of resonance wavelength and spectrum of transmitted power for mismatched DNA strands and for complementary DNA strands. For mismatched DNA strands variation is negligible whereas for complementary DNA strands is considerably countable. Proposed sensor successfully distinguishes hybridization and single nucleotide polymorphisms (SNP) by observing the variation level of resonance wavelength and spectrum of transmitted power.

  7. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    OpenAIRE

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2013-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of M...

  8. Characterization of the Arachis (Leguminosae D genome using fluorescence in situ hybridization (FISH chromosome markers and total genome DNA hybridization

    Directory of Open Access Journals (Sweden)

    Germán Robledo

    2008-01-01

    Full Text Available Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with all A and B genome taxa, supporting the special genome constitution (D genome of A. glandulifera. Genomic affinities were further investigated by dot blot hybridization of biotinylated A. glandulifera total DNA to DNA from several Arachis species, the results indicating that the D genome is positioned between the A and B genomes.

  9. PolyA-Mediated DNA Assembly on Gold Nanoparticles for Thermodynamically Favorable and Rapid Hybridization Analysis.

    Science.gov (United States)

    Zhu, Dan; Song, Ping; Shen, Juwen; Su, Shao; Chao, Jie; Aldalbahi, Ali; Zhou, Ziang; Song, Shiping; Fan, Chunhai; Zuo, Xiaolei; Tian, Yang; Wang, Lianhui; Pei, Hao

    2016-05-03

    Understanding the behavior of biomolecules on nanointerface is critical in bioanalysis, which is great challenge due to the instability and the difficulty to control the orientation and loading density of biomolecules. Here, we investigated the thermodynamics and kinetics of DNA hybridization on gold nanoparticle, with the aim to improve the efficiency and speed of DNA analysis. We achieved precise and quantitative surface control by applying a recently developed poly adenines (polyA)-based assembly strategy on gold nanoparticles (DNA-AuNPs). PolyA served as an effective anchoring block based on the preferential binding with the AuNP surface and the appended recognition block adopted an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can be systematically modulated by adjusting the length of polyA block. We found the stability of duplex on AuNP was enhanced with the increasing length of polyA block. When the length of polyA block reached to 40 bases, the thermodynamic properties were more similar to that of duplex in solution. Fast hybridization rate was observed on the diblock DNA-AuNPs and was increased along with the length of polyA block. We consider the high stability and excellent hybridization performance come from the minimization of the DNA-DNA and DNA-AuNP interactions with the use of polyA block. This study provides better understanding of the behavior of biomolecules on the nanointerface and opens new opportunities to construct high-efficiency and high-speed biosensors for DNA analysis.

  10. Self-assembly of DNA-porphyrin hybrid molecules for the creation of antimicrobial nanonetwork.

    Science.gov (United States)

    Kumari, Rina; Khan, Mohd Imran; Bhowmick, Sourav; Sinha, Kislay K; Das, Neeladri; Das, Prolay

    2017-07-01

    DNA derived well-controlled arrangement of porphyrins has emerged as promising hybrid nanostructures. Exceptional biocompatibility and DNA-directed surface addressability coupled with rich symmetry features of the porphyrin have made these hybrid nanostructures attractive candidates for potential biomedical and biotechnological applications. However, the noteworthy photophysical properties of porphyrin and related molecules when present in DNA based nanostructures are yet to be explored fully and should be a matter of intense research that may unearth a plethora of interesting applications of these nanostructures. Herein, we demonstrate the construction of novel self-assembled DNA-porphyrin hybrid nanonetworks that utilize the porphyrin core for antibacterial applications. Porphyrin derivative with four pendant NH2 groups was synthesized and conjugated with the 5'-PO4 of ss-DNA by solution phase phosphoramidation coupling reaction. The conjugation was followed by DNA hybridization mediated self-assembly to form DNA-porphyrin hybrid nanonetwork. The enhanced antimicrobial property of the nanonetwork was envisioned following light irradiation at relevant wavelength. In line with this, comparative antimicrobial activities against gram-negative (Escherichia coli BL-21) and gram-positive bacteria (Staphylococcus aureus) have been studied. Interestingly, DNA-porphyrin nanonetwork afforded highly efficient and coherent photoinduced reactive oxygen species (ROS) generation to display antimicrobial activity against Staphylococcus aureus. The escalated and coherent ROS generation from the nanonetworks was attributed to the ordered placement of the porphyrins that inhibits self-quenching. Our work points out to a good alternative for antibiotic free strategies for preservation of biological materials and other applications. Copyright © 2017. Published by Elsevier B.V.

  11. A novel method for rapid hybridization of DNA to a solid support.

    Directory of Open Access Journals (Sweden)

    Erik Pettersson

    Full Text Available Here we present a novel approach entitled Magnetic Forced Hybridization (MFH that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH approach for typing of Human Papilloma Virus (HPV. Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.

  12. A Novel Method for Rapid Hybridization of DNA to a Solid Support

    Science.gov (United States)

    Pettersson, Erik; Ahmadian, Afshin; Ståhl, Patrik L.

    2013-01-01

    Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. PMID:23950946

  13. Differential detection of pathogenic Yersinia spp. by fluorescence in situ hybridization.

    Science.gov (United States)

    Rohde, Alexander; Hammerl, Jens Andre; Appel, Bernd; Dieckmann, Ralf; Al Dahouk, Sascha

    2017-04-01

    Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogens of major medical importance, which are responsible for a considerable number of infections every year. The detection of these species still relies on cultural methods, which are slow, labour intensive and often hampered by the presence of high amounts of accompanying flora. In this study, fluorescence in situ hybridization (FISH) was used to develop a fast, sensitive and reliable alternative to detect viable bacteria in food. For this purpose, highly specific probes targeting the 16S and 23S ribosomal RNA were employed to differentially detect each of the three species. In order to enable the differentiation of single nucleotide polymorphisms (SNPs), suitable competitor oligonucleotides and locked nucleic acids (LNAs) were used. Starved cells still showed a strong signal and a direct viable count (DVC) approach combined with FISH optimized live/dead discrimination. Sensitivity of the FISH test was high and even a single cell per gram of spiked minced pork meat could be detected within a day, demonstrating the applicability to identify foodborne hazards at an early stage. In conclusion, the established FISH tests proved to be promising tools to compensate existing drawbacks of the conventional cultural detection of these important zoonotic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Anodized aluminum oxide-based capacitance sensors for the direct detection of DNA hybridization.

    Science.gov (United States)

    Kang, Bongkeun; Yeo, Unjin; Yoo, Kyung-Hwa

    2010-03-15

    We fabricated a capacitance sensor based on an anodized aluminum oxide (AAO) nanoporous structure to detect DNA hybridization. We utilized Au film deposited on the surface of the AAO membrane and Au nanowires infiltrating the nanopores as the top and bottom electrodes, respectively. When completely complementary target DNA molecules were added to the sensor-immobilized DNA molecule probes, the capacitance was reduced; with a concentration of 1pM, the capacitance decreased by approximately 10%. We measured the capacitance change for different concentrations of the target DNA solution. A linear relationship was found between the capacitance change and DNA concentration on a semi-logarithmic scale. We also investigated the possibility of detecting DNA molecules with a single-base mismatch to the probe DNA molecule. In contrast to complementary target DNA molecules, the addition of one-base mismatch DNA molecules caused no significant change in capacitance, demonstrating that DNA hybridization was detected with single nucleotide polymorphism sensitivity. (c) 2009 Elsevier B.V. All rights reserved.

  15. A microfluidic-based hybrid SPR/molecular imaging biosensor for the multiplexed detection of foodborne pathogens

    Science.gov (United States)

    Zordan, Michael D.; Grafton, Meggie M. G.; Acharya, Ghanashyam; Reece, Lisa M.; Aronson, Arthur I.; Park, Kinam; Leary, James F.

    2009-02-01

    It is important to screen our food supply for pathogen contaminations. Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food where it is produced or distributed. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface due to changes in refractive index that occur upon binding. It can be used for label-free detection of the presence of potential pathogens. Simultaneous fluorescence molecular imaging on the other side of the biochip can be used to ascertain pathogen status or functional state which may affect its potential danger to humans or animals. We are designing and testing hybrid microfluidic biochips to detect multiple pathogens using a combination of SPRI and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a peptide targeting a specific pathogen. This peptide biosensor array is enclosed by a PDMS microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. An SPR image is taken from the bottom of the biochip. Image analysis is used to quantify the amount of pathogen (both live and dead) bound to each spot. Since PDMS is very transmissive to visible light, an epi-fluorescence image is taken from the top of the biochip. Fluorescence imaging determines the live:dead ratio of each pathogen using an inexpensive SYTO 9(R)-Propidium Iodide assay. The volume of sample that the biochip can analyze is small, so possible pathogens are pre-concentrated using immunomagnetic separation. Functionalized magnetic particles are bound to pathogens present in the sample, and a magnet is used to separate them from the bulk fluid.

  16. Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array.

    Science.gov (United States)

    Devault, Alison M; McLoughlin, Kevin; Jaing, Crystal; Gardner, Shea; Porter, Teresita M; Enk, Jacob M; Thissen, James; Allen, Jonathan; Borucki, Monica; DeWitte, Sharon N; Dhody, Anna N; Poinar, Hendrik N

    2014-03-06

    Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis ("Black Death" plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

  17. Direct Measurement of Single-Molecule DNA Hybridization Dynamics with Single-Base Resolution.

    Science.gov (United States)

    He, Gen; Li, Jie; Ci, Haina; Qi, Chuanmin; Guo, Xuefeng

    2016-07-25

    Herein, we report label-free detection of single-molecule DNA hybridization dynamics with single-base resolution. By using an electronic circuit based on point-decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal-to-noise ratio and bandwidth. These measurements reveal two-level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base-by-base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base-pair level. This measurement capability promises a label-free single-molecule approach to probe biomolecular interactions with fast dynamics.

  18. Hybridization detection of enzyme-labeled DNA at electrically heated electrodes.

    Science.gov (United States)

    Walter, Anne; Surkus, Annette-Enrica; Flechsig, Gerd-Uwe

    2013-04-01

    In this report we describe an electrochemical DNA hybridization sensor approach, in which signal amplification is achieved using heated electrodes together with an enzyme as DNA-label. On the surface of the heatable low temperature co-fired ceramic (LTCC) gold electrode, an immobilized thiolated capture probe was hybridized with a biotinylated target using alkaline phosphatase (SA-ALP) as reporter molecule. The enzyme label converted the redox-inactive substrate 1-naphthyl phosphate (NAP) into the redox-active 1-naphthol voltammetrically determined at the modified gold LTCC electrode. During the measurement only the electrode was heated leaving the bulk solution at ambient temperature. Elevated temperature during detection led to increased enzyme activity and enhanced analytical signals for DNA hybridization detection. The limit of detection at 53 °C electrode temperature was 1.2 nmol/L.

  19. Multilayer DNA Origami Packed on Hexagonal and Hybrid Lattices

    DEFF Research Database (Denmark)

    Ke, Yonggang; Voigt, Niels Vinther; Shih, William M.

    2012-01-01

    “Scaffolded DNA origami” has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry. H...

  20. Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

    Institute of Scientific and Technical Information of China (English)

    Rongzhi Wang; Jiadong Huang; Wei Zhang; Guangmei Lin; Junwei Lian; Libin Jiang; Hongcong Lin; Songfa Wang; Shihua Wang

    2011-01-01

    In this paper,we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains,using multiplex PCR and DNA-DNA hybridization.Multiplex PCR was used to simultaneously amplify three diagnostic genes(tlh,tdh and fia)that serve as molecular markers of V.parahaemolyticus.Biotinylated PCR products were hybridized to primers immobilized on a microarray,and detected by chemiluminesce with avidin-conjugated alkaline phosphatase.With this method,forty-five samples were tested.Eight known virulent strains (tlh+/tdh+/fia+)and four known avirulent strains(tlh+/tdh-/fla+)of the V.parahaemolyttcus were successtuny aetectea,ana no non-spectnc hybridization and cross-hybridization reaction were found from fifteen closely-related strains(tin-/tdh-/fta+)or the Vibrio spp.In addition,all the other eighteen strains of non-Vibrio bacteria(tlh-/tdh-/fla-)gave negative results.The DNA microarray successfully distinguished V.parahaemolyticus from other Vibrio spp.The results demonstrated that this was an efficient and robust method for identifying virulent strains of V.parahaemolyticus.

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

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

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

  2. DNA mutations mediate microevolution between host-adapted forms of the pathogenic fungus Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Denise A Magditch

    Full Text Available The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a "switch" from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.

  3. A cyclodextrin host-guest recognition approach to a label-free electrochemical DNA hybridization biosensor.

    Science.gov (United States)

    Abbaspour, Abdolkarim; Noori, Abolhassan

    2012-04-21

    A novel label-free electrochemical DNA hybridization biosensor using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified screen printed electrode (CD/PNAANI/CNT/SPE) has been developed. The proposed DNA hybridization biosensor relies on the intrinsic oxidation signals of guanine (G) and adenine (A) from single-stranded DNA entered into the cyclodextrin (CD) cavity. Due to the binding of G and A bases to complementary cytosine and thymine bases in dsDNA, the signals obtained for ssDNA were much higher than that of dsDNA. The synergistic effect of the multi-walled carbon nanotubes provides a significantly enhanced voltammetric signal, and the CD encapsulation effect makes anodic peaks of G and A shift to less positive potentials than that at the bare SPE. The peak heights of G and A signals are dependent on both the number of the respective bases in oligonucleotides and the concentration of the target DNA sequences. Hybridization of complementary strands was monitored through the measurements of oxidation signal of purine bases, which enabled the detection of target sequences from 0.01 to 1.02 nmol μl(-1) with the detection limit of target DNA as low as 5.0 pmol μl(-1) (S/N = 3). Implementation of label-free and homogeneous electrochemical hybridization detection constitutes an important step toward low-cost, simple, highly sensitive and accurate DNA assay. Discrimination between complementary, noncomplementary, and two-base mismatch targets was easily accomplished using the proposed electrode.

  4. 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.

  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. Programmed protection of foreign DNA from restriction allows pathogenicity island exchange during pneumococcal transformation.

    Directory of Open Access Journals (Sweden)

    Calum Johnston

    2013-02-01

    Full Text Available In bacteria, transformation and restriction-modification (R-M systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the

  7. A novel SERRS sandwich-hybridization assay to detect specific DNA target.

    Directory of Open Access Journals (Sweden)

    Cécile Feuillie

    Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

  8. Magnetoresistive sensors for measurements of DNA hybridization kinetics – effect of TINA modifications

    Science.gov (United States)

    Rizzi, G.; Dufva, M.; Hansen, M. F.

    2017-01-01

    We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current. A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5′ end (1 × TINA) or at both the 5′ and 3′ ends (2 × TINA) were compared. TINA modifications were found to provide a relative decrease of koff by a factor of 6-20 at temperatures from 57.5 °C to 60 °C. The values of kon were generally in the range between 0.5-2 × 105 M−1s−1 and showed lower values for the unmodified probe than for the TINA modified probes. The observations correlated well with measured melting temperatures of the DNA hybrids. PMID:28167835

  9. 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.

  10. 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.

  11. A novel gold nanoparticle-DNA aptamer-based plasmonic chip for rapid and sensitive detection of bacterial pathogens

    DEFF Research Database (Denmark)

    Sun, Yi; Phuoc Long, Truong; Wolff, Anders

    2016-01-01

    Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers...

  12. Preparation of bio-deep eutectic solvent triggered cephalopod shaped silver chloride-DNA hybrid material having antibacterial and bactericidal activity.

    Science.gov (United States)

    Bhatt, Jitkumar; Mondal, Dibyendu; Bhojani, Gopal; Chatterjee, Shruti; Prasad, Kamalesh

    2015-11-01

    2.5% w/w DNA (Salmon testes) was solubilized in a bio-deep eutectic solvent [(bio-DES), obtained by the complexation of choline chloride and ethylene glycol at 1:2 molar ratio] containing 1% w/w of silver chloride (AgCl) to yield a AgCl decorated DNA based hybrid material. Concentration dependent formation of AgCl crystals in the DES was observed and upon interaction with DNA it gave formation of a cephalopod shaped hybrid material. DNA was found to maintain its chemical and structural stability in the material. Further, AgCl microstructures were found to have orderly self assembled on the DNA helices indicating the electrostatic interaction between Ag(+) and phosphate side chain of DNA as a driving force for the formation of the material with ordered microstructural distribution of AgCl. Furthermore, the functionalized material exhibited excellent antibacterial and bactericidal activity against both Gram negative and Gram positive pathogenic bacteria.

  13. DNA microarray-based detection of multiple pathogens: Mycoplasma spp. and Chlamydia spp.

    Science.gov (United States)

    Schnee, Christiane; Sachse, Konrad

    2015-01-01

    Rapid detection of slow-growing or non-culturable microorganisms, such as Mycoplasma spp. and Chlamydia spp., is still a challenge to diagnosticians in the veterinary field. In addition, as epidemiological evidence on the frequency of mixed infections involving two and more bacterial species has been emerging, detection methods allowing simultaneous identification of different pathogens are required. In the present chapter, we describe DNA microarray-based procedures for the detection of 83 Mollicutes species (Mycoplasma assay) and 11 Chlamydia spp. (Chlamydia assay). The assays are suitable for use in a routine diagnostic environment, as well as in microbiological research.

  14. Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes.

    Science.gov (United States)

    Kormuth, Karen A; Woolford, John L; Armitage, Bruce A

    2016-03-29

    Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

  15. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    OpenAIRE

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Roy; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    Abstract: The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to e...

  16. A new method for identification of Trichomonas vaginalis by fluorescent DNA in situ hybridization.

    OpenAIRE

    Muresu, R; Rubino, S.; Rizzu, P.; Baldini, A.; Colombo, M; Cappuccinelli, P.

    1994-01-01

    The protozoan flagellate Trichomonas vaginalis is responsible for human trichomoniasis, one of the most widespread sexually transmitted diseases in the world. Several methods are currently used for laboratory diagnosis, including direct microscopic observation, cell culture, immunological techniques, and more recently, DNA probing and gene amplification. This report describes an in situ hybridization technique with specific DNA probes labeled with either biotin, rhodamine, or fluorescein for ...

  17. Fusarium foetens, a new species pathogenic to begonia elatior hybrids (Begonia x hiemalis) and the sister taxon of the Fusarium oxysporum species complex.

    Science.gov (United States)

    Schroers, H-J; Baayen, R P; Meffert, J P; de Gruyter, J; Hooftman, M; O'Donnell, K

    2004-01-01

    A new disease recently was discovered in begonia elatior hybrid (Begonia × hiemalis) nurseries in The Netherlands. Diseased plants showed a combination of basal rot, vein yellowing and wilting and the base of collapsing plants was covered by unusually large masses of Fusarium macroconidia. A species of Fusarium was isolated consistently from the discolored veins of leaves and stems. It differed morphologically from F. begoniae, a known agent of begonia flower, leaf and stem blight. The Fusarium species resembled members of the F. oxysporum species complex in producing short monophialides on the aerial mycelium and abundant chlamydospores. Other phenotypic characters such as polyphialides formed occasionally in at least some strains, relatively long monophialides intermingled with the short monophialides formed on the aerial mycelium, distinct sporodochial conidiomata, and distinct pungent colony odor distinguished it from the F. oxysporum species complex. Phylogenetic analyses of partial sequences of the mitochondrial small subunit of the ribosomal DNA (mtSSU rDNA), nuclear translation elongation factor 1α (EF-1α) and β-tubulin gene exons and introns indicate that the Fusarium species represents a sister group of the F. oxysporum species complex. Begonia × hiemalis cultivars Bazan, Bellona and Netja Dark proved to be highly susceptible to the new species. Inoculated plants developed tracheomycosis within 4 wk, and most died within 8 wk. The new taxon was not pathogenic to Euphorbia pulcherrima, Impatiens walleriana and Saintpaulia ionantha that commonly are grown in nurseries along with B. × hiemalis. Inoculated plants of Cyclamen persicum did not develop the disease but had discolored vessels from which the inoculated fungus was isolated. Given that the newly discovered begonia pathogen is distinct in pathogenicity, morphology and phylogeny from other fusaria, it is described here as a new species, Fusarium foetens.

  18. Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Genomic DNA polymorphisms are very useful for tracing genetic traits and studying biological diversity among species. Here, we present a method we call the "diversity suppression-subtractive hybridization array" for effectively profiling genomic DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization (SSH) to establish a subtracted gDNA library,from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomic DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphic fragments of pairwise comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array is a highly effective platform for profiling genomic DNA polymorphisms and dendrograms.

  19. Submillimetre Network Formation by Light-induced Hybridization of Zeptomole-level DNA

    Science.gov (United States)

    Iida, Takuya; Nishimura, Yushi; Tamura, Mamoru; Nishida, Keisuke; Ito, Syoji; Tokonami, Shiho

    2016-12-01

    Macroscopic unique self-assembled structures are produced via double-stranded DNA formation (hybridization) as a specific binding essential in biological systems. However, a large amount of complementary DNA molecules are usually required to form an optically observable structure via natural hybridization, and the detection of small amounts of DNA less than femtomole requires complex and time-consuming procedures. Here, we demonstrate the laser-induced acceleration of hybridization between zeptomole-level DNA and DNA-modified nanoparticles (NPs), resulting in the assembly of a submillimetre network-like structure at the desired position with a dramatic spectral modulation within several minutes. The gradual enhancement of light-induced force and convection facilitated the two-dimensional network growth near the air-liquid interface with optical and fluidic symmetry breakdown. The simultaneous microscope observation and local spectroscopy revealed that the assembling process and spectral change are sensitive to the DNA sequence. Our findings establish innovative guiding principles for facile bottom-up production via various biomolecular recognition events.

  20. DNA Hybridization Detection Based on Resonance Frequency Readout in Graphene on Au SPR Biosensor

    Directory of Open Access Journals (Sweden)

    Md. Biplob Hossain

    2016-01-01

    Full Text Available This paper demonstrates a numerical modeling of surface plasmon resonance (SPR biosensor for detecting DNA hybridization by recording the resonance frequency characteristics (RFC. The proposed sensor is designed based on graphene material as biomolecular recognition elements (BRE and the sharp SPR curve of gold (Au. Numerical analysis shows that the variation of RFC for mismatched DNA strands is quiet negligible whereas that for complementary DNA strands is considerably countable. Here, graphene is used to perform faster immobilization between target DNA and probe DNA. The usage of graphene also changes the RFC that ensure hybridization of DNA event by utilizing its optochemical property. In addition, proposed sensor successfully distinguishes between hybridization and single-nucleotide polymorphisms (SNP by observing the variation level of RFC and maximum transmittance. Therefore, the proposed frequency readout based SPR sensor could potentially open a new window of detection for biomolecular interactions. We also highlight the advantage of using graphene sublayer by performing the sensitivity analysis. Sandwiching of each graphene sublayer enhances 95% sensitivity comparing with conventional SPR sensor.

  1. 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.

  2. [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.

  3. Modified Genetic Algorithm for DNA Sequence Assembly by Shotgun and Hybridization Sequencing Techniques

    Directory of Open Access Journals (Sweden)

    Prof.Narayan Kumar Sahu

    2012-09-01

    Full Text Available Since the advent of rapid DNA sequencing methods in 1976, scientists have had the problem of inferring DNA sequences from sequenced fragments. Shotgun sequencing is a well-established biological and computational method used in practice. Many conventional algorithms for shotgun sequencing are based on the notion of pair wise fragment overlap. While shotgun sequencing infers a DNA sequence given the sequences of overlapping fragments, a recent and complementary method, called sequencing by hybridization (SBH, infers a DNA sequence given the set of oligomers that represents all sub words of some fixed length, k. In this paper, we propose a new computer algorithm for DNA sequence assembly that combines in a novel way the techniques of both shotgun and SBH methods. Based on our preliminary investigations, the algorithm promises- to be very fast and practical for DNA sequence assembly [1].

  4. Nanomaterial-Assisted Signal Enhancement of Hybridization for DNA Biosensors: A Review

    Directory of Open Access Journals (Sweden)

    Minqiang Li

    2009-09-01

    Full Text Available Detection of DNA sequences has received broad attention due to its potential applications in a variety of fields. As sensitivity of DNA biosensors is determined by signal variation of hybridization events, the signal enhancement is of great significance for improving the sensitivity in DNA detection, which still remains a great challenge. Nanomaterials, which possess some unique chemical and physical properties caused by nanoscale effects, provide a new opportunity for developing novel nanomaterial-based signal-enhancers for DNA biosensors. In this review, recent progress concerning this field, including some newly-developed signal enhancement approaches using quantum-dots, carbon nanotubes and their composites reported by our group and other researchers are comprehensively summarized. Reports on signal enhancement of DNA biosensors by non-nanomaterials, such as enzymes and polymer reagents, are also reviewed for comparison. Furthermore, the prospects for developing DNA biosensors using nanomaterials as signal-enhancers in future are also indicated.

  5. Immobilization and Hybridization of cDNA Arrays on the Glass Surface

    Institute of Scientific and Technical Information of China (English)

    谢文章; 孙宝全; 周玉祥; 程京

    2002-01-01

    DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules in one sample. The efficiency of the array mainly depends on the sequence of the capture probes and the way in which they are attached to the support. Coupling process needs to be quick, reproducible and compatible with automatic spotting devices dispensing tiny drops of liquid on the glass surface. Coupling strategies were evaluated in light of grafting DNA onto the glass surface. The results indicate that attaching unmodified DNA to a poly-L-lysine coated glass surface is a preferred method in fabricating DNA microarrays. In this paper, both the coupling procedure and the hybridization efficiency have been optimized. The unmodified double-stranded DNA in high salt solution being spotted onto the poly-L-lysine coated glass surface enhances the adsorption of DNA onto the poly-L-lysine layer.

  6. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing

    Science.gov (United States)

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R.

    1993-06-01

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

  7. Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja;

    2002-01-01

    It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid...... DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens...... whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non...

  8. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  9. Label-free detection of DNA hybridization using InAs μ-Hall sensors

    Science.gov (United States)

    Aledealat, Khaled; Hira, S.; Chen, K.; Strouse, G. F.; Chase, P. B.; Xiong, P.; von Molnar, S.; Mihajlovic, G.; Field, M.; Sullivan, G.

    2010-03-01

    We present results on label-free detection of DNA hybridization using InAs μ-Hall sensors. The μ-Hall sensor consisted of six 1-μm Hall crosses defined on an InAs quantum well substrate. The sensor was then covered with sputter-deposited SiO2 and Au pads were patterned on top of some of the Hall crosses. Thiolated ssDNA strands that are complementary to one end of the target ssDNA were assembled on the Au pads and the rest of the device platform was passivated with PEG-silane. Biotinylated and fluorescently-tagged complementary ssDNA to the other end of the target ssDNA were labeled with commercial streptavidin-coated 350 nm superparamagnetic beads. Labeled ssDNA were found to assemble selectively onto the Au pads after mixing with the target ssDNA, indicating successful hybridization of the three ssDNA sequences. The presence of the assembled beads was successfully detected via the Hall sensor and confirmed using laser scanning confocal microscopy. This work was supported by NIH NIGMS GM079592.

  10. Preparation of Luminol-doped Nanoparticle and Its Application in DNA Hybridization Analysis

    Institute of Scientific and Technical Information of China (English)

    QIAN,Ke-Jun(钱柯君); ZHANG,Ling(张玲); YANG,Min-Li(杨敏丽); HE,Pin-Gang(何品刚); FANG,Yu-Zhi(方禹之)

    2004-01-01

    The dye-doped silica nanoparticles can be used as nanobiosensors that are able to recognize and detect specific DNA sequence. In this paper, spherical nanosized luminol/SiO2 composite particles have been synthesized with reverse micells via hydrolysis of tetraethyl orthosilicate (TEOS) in the microemulsion. The nanoparticles were modified with chitosan and used to label DNA, forming the DNA probe which was used to hybridize with target DNA immobilized on a PPy modified Pt electrode. The hybridization events were evaluated by electrogenerated chemiluminescence (ECL) measurements and only the complementary sequence could form a double-stranded DNA (dsDNA) with DNA probe and give strong ECL signals. A three base mismatch sequence and a non-complementary sequence had almost negligible responses. Due to the large number of luminol molecules inside silica nanoparticles, the assay allows detection at levels as low as 2.0 × 10- 12 mol/L of the target DNA. The intensity of ECL was linearly related to the concentration of the complementary sequence in the range of 5.0 × 10-12-1.0 ×10-9 mol/L.

  11. Genome-scale DNA sequence recognition by hybridization to short oligomers.

    Science.gov (United States)

    Milosavljević, A; Savković, S; Crkvenjakov, R; Salbego, D; Serrato, H; Kreuzer, H; Gemmell, A; Batus, S; Grujić, D; Carnahan, S; Tepavcević, J

    1996-01-01

    Recently developed hybridization technology (Drmanac et al. 1994) enables economical large-scale detection of short oligomers within DNA fragments. The newly developed recognition method (Milosavljević 1995b) enables comparison of lists of oligomers detected within DNA fragments against known DNA sequences. We here describe an experiment involving a set of 4,513 distinct genomic E.coli clones of average length 2kb, each hybridized with 636 randomly selected short oligomer probes. High hybridization signal with a particular probe was used as an indication of the presence of a complementary oligomer in the particular clone. For each clone, a list of oligomers with highest hybridization signals was compiled. The database consisting of 4,513 oligomer lists was then searched using known E.coli sequences as queries in an attempt to identify the clones that match the query sequence. Out of a total of 11 clones that were recognized at highest significance level by our method, 8 were single-pass sequenced from both ends. The single-pass sequenced ends were then compared against the query sequences. The sequence comparisons confirmed 7 out of the total of 8 examined recognitions. This experiment represents the first successful example of genome-scale sequence recognition based on hybridization data.

  12. Microbial pathogens trigger host DNA double-strand breaks whose abundance is reduced by plant defense responses.

    Directory of Open Access Journals (Sweden)

    Junqi Song

    2014-04-01

    Full Text Available Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.

  13. 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.

  14. Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization.

    Science.gov (United States)

    Kalogianni, Despina P; Boutsika, Lemonia M; Kouremenou, Panagiota G; Christopoulos, Theodore K; Ioannou, Penelope C

    2011-05-01

    Presently, there is a growing interest in the development of lateral flow devices for nucleic acid analysis that enable visual detection of the target sequence (analyte) while eliminating several steps required for pipetting, incubation, and washing out the excess of reactants. In this paper, we present, for the first time, lateral flow tests exploiting oligonucleotide-functionalized and antibody-functionalized carbon nanoparticles (carbon nano-strings, CBNS) as reporters that enable confirmation of the target DNA sequence by hybridization. The CBNS reporters were applied to (a) the detection of PCR products and (b) visual genotyping of single nucleotide polymorphisms in human genomic DNA. Biotinylated PCR product was hybridized with a dA-tailed probe. In one assay configuration, the hybrid is captured at the test zone of the strip by immobilized streptavidin and detected by (dT)(30)-CBNS. In a second configuration, the hybrids are captured from immobilized (dA) strands and detected by antibiotin-CBNS. As low as 2.5 fmol of amplified DNA can be detected. For visual genotyping, allele-specific primers with a 5' oligo(dA) segment are extended by DNA polymerase with a concomitant incorporation of biotin moieties. Extension products are detected either by (dT)(30)-CBNS or by antibiotin-CBNS. Only three cycles of extension reaction are sufficient for detection. No purification of the PCR products or the extension product is required.

  15. DNA copy number aberrations in breast cancer by array comparative genomic hybridization

    DEFF Research Database (Denmark)

    Li, J.; Wang, K.; Li, S.;

    2009-01-01

    Array comparative genomic hybridization (CGH) has been popularly used for analyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb resolution bacterial artificial chromosome CGH arrays. A number of h...

  16. Active microelectronic array system for DNA hybridization, genotyping and pharmacogenomic applications.

    Science.gov (United States)

    Sosnowski, Ron; Heller, Michael J; Tu, Eugene; Forster, Anita H; Radtkey, Ray

    2002-12-01

    Microelectronic arrays have been developed for DNA hybridization analysis of point mutations, single nucleotide polymorphisms, short tandem repeats and gene expression. In addition to a variety of molecular biology and genomic research applications, such devices will also be used for infectious disease detection, genetic and cancer diagnostics, and pharmacogenomic applications. These microelectronic array devices are able to produce defined electric fields on their surfaces that allow charged molecules and other entities to be transported to or from any test site or micro-location on the planar surface of the device. These molecules and entities include DNA, RNA, proteins, enzymes, antibodies and cells. Electronic-based molecule addressing and hybridization can then be carried out, where the electric field is now used to greatly accelerate the hybridization reactions that occur on the selected test sites. When reversed, the electric field can be used to provide an additional parameter for improved hybridization. Special low-conductance buffers have been developed that provide for the rapid transport of the DNA molecules and facilitate the electronic hybridization reactions under conditions that do not support hybridization. Important to the device function is the permeation layer that overcoats the underlying microelectrodes. Generally composed of a porous hydrogel material impregnated with attachment chemistry, this permeation layer prevents the destruction of analytes at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization and affinity reactions, and serves as a support structure for attaching DNA probes and other molecules to the array. The microelectronic chip or array device is incorporated into a cartridge package (NanoChip trade mark cartridge) that provides the electronic, optical, and fluidic interfacing. A complete instrument system (NanoChip trade mark Molecular Biology Workstation

  17. CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

    Science.gov (United States)

    Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

    2016-04-01

    To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

  18. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. Copyright

  19. Harnessing DNA Synthesis to Develop Rapid Responses to Emerging and Pandemic Pathogens

    Directory of Open Access Journals (Sweden)

    Lisa M. Runco

    2011-01-01

    Full Text Available Given the interconnected nature of our world today, emerging pathogens and pandemic outbreaks are an ever-growing threat to the health and economic stability of the global community. This is evident by the recent 2009 Influenza A (H1N1 pandemic, the SARS outbreak, as well as the ever-present threat of global bioterrorism. Fortunately, the biomedical community has been able to rapidly generate sequence data so these pathogens can be readily identified. To date, however, the utilization of this sequence data to rapidly produce relevant experimental results or actionable treatments is lagging in spite of obtained sequence data. Thus, a pathogenic threat that has emerged and/or developed into a pandemic can be rapidly identified; however, translating this identification into a targeted therapeutic or treatment that is rapidly available has not yet materialized. This commentary suggests that the growing technology of DNA synthesis should be fully implemented as a means to rapidly generate in vivo data and possibly actionable therapeutics soon after sequence data becomes available.

  20. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    Science.gov (United States)

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2014-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

  1. Numerical investigation of magnetic sensor for DNA hybridization detection using planar transformer

    Directory of Open Access Journals (Sweden)

    Sayyed M. Azimi

    2007-12-01

    Full Text Available This paper introduces a sensor for detection of DNA hybridization and investigates its performance by means of computer simulation. A planar transformer with spiral windings is proposed for hybridization detection. In order to detect the occurrence of hybridization, single strand target DNA’s are tagged with magnetic beads. Target DNA’s are then exposed to known single strand probe DNA’s which are immobilized on the surface of a functionalized layer in the proximity of the sensor. The primary winding of the transformer is driven by an AC current source. The voltage at the secondary winding is used for detection. Once the hybridization is occurred, a layer of magnetic material is formed and the coupling between the windings is varied. These variations are reflected into the detecting output voltage. The magnitude of the output voltage is numerically calculated in terms of geometrical and physical parameters and the parameter values resulting in maximum response are derived.

  2. Direct detection of expanded trinucleotide repeats using PCR and DNA hybridization techniques

    Energy Technology Data Exchange (ETDEWEB)

    Petronis, A.; Tatuch, Y.; Klempan, T.A.; Kennedy, J.L. [Hospital for Sick Children, Toronto (Canada)] [and others

    1996-02-16

    Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG){sub n} expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG){sub n}/(CAG){sub n} repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA. 33 refs., 4 figs.

  3. PHOTOPROBER® Biotin: An Alternative Method for Labeling Archival DNA for Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Dirk Korinth

    2004-01-01

    Full Text Available Comparative genomic hybridization (CGH represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin‐fixed, paraffin‐embedded (FFPE tissue. A well‐known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non‐enzymatic, PHOTOPROBE® biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE® samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE® biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

  4. SOME FEATURES OF HYDROLYSIS OF THE HYBRID B-Z-FORM DNA BY SERRATIA MARCESCENS NUCLEASE

    Directory of Open Access Journals (Sweden)

    Maria Filimonova

    2014-01-01

    Full Text Available Highly polymerized herring testis DNA of the random nucleotide sequence was used as a model of natural substrate to study some features of hydrolysis of the hybrid B-Z form with Serratia marcescens nuclease. The hybrid B-Z-form was formed upon addition of 1.15 M MgSO4 and 0.421 mM Co(NH36Cl3. The DNA transition from the right handed B-form to the hybrid B-Z-form caused a decrease in Vmax of DNA cleavage with the nuclease. The diminishing Vmax was consistent with diminishing values of Km and Kcat. The binding of Mg2+ or Co(NH363+ to highly polymerized DNA caused correspondingly about 80-or 7-fold decrease in Km and more than 1600 or 600 decrease in Kcat compared with that of Mg-DNA complex of B-form.

  5. Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit.

    Science.gov (United States)

    Yanagihara, Katsunori; Kitagawa, Yuko; Tomonaga, Masao; Tsukasaki, Kunihiro; Kohno, Shigeru; Seki, Masafumi; Sugimoto, Hisashi; Shimazu, Takeshi; Tasaki, Osamu; Matsushima, Asako; Ikeda, Yasuo; Okamoto, Shinichiro; Aikawa, Naoki; Hori, Shingo; Obara, Hideaki; Ishizaka, Akitoshi; Hasegawa, Naoki; Takeda, Junzo; Kamihira, Shimeru; Sugahara, Kazuyuki; Asari, Seishi; Murata, Mitsuru; Kobayashi, Yoshio; Ginba, Hiroyuki; Sumiyama, Yoshinobu; Kitajima, Masaki

    2010-01-01

    Sepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. Conventional methods take three or more days for final pathogen identification and antimicrobial susceptibility testing. We organized a prospective observational multicenter study in three study sites to evaluate the diagnostic accuracy and potential clinical utility of the SeptiFast system, a multiplex pathogen detection system used in the clinical setting to support early diagnosis of bloodstream infections. A total of 212 patients, suspected of having systemic inflammatory response syndrome (SIRS) caused by bacterial or fungal infection, were enrolled in the study. From these patients, 407 blood samples were taken and blood culture analysis was performed to identify pathogens. Whole blood was also collected for DNA Detection Kit analysis immediately after its collection for blood culture. The results of the DNA Detection Kit, blood culture and other culture tests were compared. The chosen antimicrobial treatment in patients whose samples tested positive in the DNA Detection Kit and/or blood culture analysis was examined to evaluate the effect of concomitant antibiotic exposure on the results of these analyses. SeptiFast analysis gave a positive result for 55 samples, while 43 samples were positive in blood culture analysis. The DNA Detection Kit identified a pathogen in 11.3% (45/400) of the samples, compared to 8.0% (32/400) by blood culture analysis. Twenty-three pathogens were detected by SeptiFast only; conversely, this system missed five episodes of clinically significant bacteremia (Methicillin-resistant Staphylococcus aureus (MRSA), 2; Pseudomonas aeruginosa, 1; Klebsiella spp, 1; Enterococcus faecium, 1). The number of samples that tested positive was significantly increased by combining the result of the blood culture analysis with those of the DNA Detection Kit analysis (P = 0.01). Among antibiotic pre-treated patients (prevalence, 72%), Septi

  6. DNA Barcoding for Efficient Species- and Pathovar-Level Identification of the Quarantine Plant Pathogen Xanthomonas

    Science.gov (United States)

    Tian, Qian; Zhao, Wenjun; Lu, Songyu; Zhu, Shuifang; Li, Shidong

    2016-01-01

    Genus Xanthomonas comprises many economically important plant pathogens that affect a wide range of hosts. Indeed, fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria for imports in China. To date, however, a rapid and accurate method capable of identifying all of the quarantine species/pathovars has yet to be developed. In this study, we therefore evaluated the capacity of DNA barcoding as a digital identification method for discriminating quarantine species/pathovars of Xanthomonas. For these analyses, 327 isolates, representing 45 Xanthomonas species/pathovars, as well as five additional species/pathovars from GenBank (50 species/pathovars total), were utilized to test the efficacy of four DNA barcode candidate genes (16S rRNA gene, cpn60, gyrB, and avrBs2). Of these candidate genes, cpn60 displayed the highest rate of PCR amplification and sequencing success. The tree-building (Neighbor-joining), ‘best close match’, and barcode gap methods were subsequently employed to assess the species- and pathovar-level resolution of each gene. Notably, all isolates of each quarantine species/pathovars formed a monophyletic group in the neighbor-joining tree constructed using the cpn60 sequences. Moreover, cpn60 also demonstrated the most satisfactory results in both barcoding gap analysis and the ‘best close match’ test. Thus, compared with the other markers tested, cpn60 proved to be a powerful DNA barcode, providing a reliable and effective means for the species- and pathovar-level identification of the quarantine plant pathogen Xanthomonas. PMID:27861494

  7. Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis.

    Science.gov (United States)

    Bletz, M C; Rebollar, E A; Harris, R N

    2015-02-10

    Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.

  8. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH in buccal cells

    Directory of Open Access Journals (Sweden)

    E. I. Cortés-Gutiérrez

    2012-12-01

    Full Text Available DNA breakage detection-fluorescence in situ hybridization (DBD-FISH is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91. In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  9. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in buccal cells.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Fernández, J L; López-Fernández, C; Gosálvez, J

    2012-12-28

    DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  10. Sterically controlled docking of gold nanoparticles on ferritin surface by DNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, B; Uenuma, M; Iwahori, K; Okamoto, N; Naito, M; Ishikawa, Y; Uraoka, Y; Yamashita, I [Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192 (Japan)

    2011-07-08

    Novel assemblies of DNA-functionalized gold nanoparticles (DNA-GNPs) have received considerable interest due to their fascinating properties which are desired for various detection applications. In this study, we present innovative GNP assemblies which have a cage-shaped protein ferritin in the center, and discrete GNPs sterically surrounding the central ferritin. These assemblies were constructed by hybridizing DNA-GNP to chemically DNA-modified ferritin, which has a hollow cavity or an iron NP core. Subsequent gel electrophoresis purification and transmission electron microscopy observation showed that ferritin/DNA/GNP assemblies were successfully constructed and can be isolated as independent functional units, which can be used to investigate not only the interaction between the GNPs of complicated GNP clusters but also the interaction between the GNPs and the internalized NP.

  11. A microfluidic-based electrochemical biochip for label-free DNA hybridization analysis.

    Science.gov (United States)

    Ben-Yoav, Hadar; Dykstra, Peter H; Gordonov, Tanya; Bentley, William E; Ghodssi, Reza

    2014-09-10

    Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

  12. Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures.

    Science.gov (United States)

    Kim, Wonyong; Park, Hee Kuk; Thanh, Hien Dang; Lee, Bo-Young; Shin, Jong Wook; Shin, Hyoung-Shik

    2011-12-01

    Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.

  13. Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lianquan; LIU Dengcai; YAN Zehong; ZHENG Youliang

    2005-01-01

    Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n = 42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.

  14. Human cDNA mapping using fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  15. Atomistic details of the molecular recognition of DNA-RNA hybrid duplex by ribonuclease H enzyme

    Indian Academy of Sciences (India)

    Gorle Suresh; U Deva Priyakumar

    2015-10-01

    Bacillus halodurans (ℎ) ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and is a prototypical member of a large family of enzymes that use two-metal ion (Mg2+ or Mn2+) catalysis to cleave nucleic acids. Long timescale molecular dynamics simulations have been performed on the ℎRNase H-DNA-RNA hybrid complex and the respective monomers to understand the recognition mechanism, conformational preorganization, active site dynamics and energetics involved in the complex formation. Several structural and energetic analyses were performed and significant structural changes are observed in enzyme and hybrid duplex during complex formation. Hybrid molecule binding to RNase H enzyme leads to conformational changes in the DNA strand. The ability of the DNA strand in the hybrid duplex to sample conformations corresponding to typical A- and B-type nucleic acids and the characteristic minor groove width-seem to be crucial for efficient binding. Sugar moieties in certain positions interacting with the protein structure undergo notable conformational transitions. The water coordination and arrangement around the metal ions in active site region are quite stable, suggesting their important role in enzymatic catalysis. Details of key interactions found at the interface of enzyme-nucleic acid complex that are responsible for its stability are discussed.

  16. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    Science.gov (United States)

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

  17. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

    Science.gov (United States)

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

  18. Ensembles of nanoelectrodes modified with gold nanoparticles: characterization and application to DNA-hybridization detection.

    Science.gov (United States)

    Silvestrini, Morena; Ugo, Paolo

    2013-01-01

    A new method to increase the active area (A(act)) of nanoelectrode ensembles (NEEs) is described. To this aim, gold nanoparticles (AuNPs) are immobilized onto the surface of NEEs using cysteamine as a cross-linker able to bind the AuNPs to the heads of the nanoelectrodes to obtain the so-called AuNPs-NEEs. The analysis of the cyclic voltammograms recorded in pure supporting electrolyte showed that the presence of the nanoparticles reflects in an, approximately, ten-times increase in the electrochemically active area of the ensemble. The measurement of the amount of electroactive polyoxometalates, which can be adsorbed on the gold surface of NEEs vs. AuNPs-NEEs, confirmed a significant increase of active area for the latter. These evidences indicate that there is a good electronic connection between the AuNPs and the underlying nanoelectrodes. The possibility to exploit AuNPs-NEEs for biosensing application was tested for the case of DNA-hybridization detection. After immobilization on the gold surface of AuNPs-NEEs of a thiolated single-stranded DNA, the hybridization with complementary sequences labeled with glucose oxidase (GOx) was performed. The detection of the hybridization was achieved by adding to the electrolyte solution the GOx substrate (i.e., glucose) and a suitable redox mediator, namely the (ferrocenylmethyl) trimethylammonium (FA(+)) cation; when the hybridization occurs, an electrocatalytic increase of the oxidation current of FA(+) is recorded. Comparison of electrocatalytic current recorded at DNA modified NEEs and AuNPs-NEEs indicate, for the latter, a significant increase in sensitivity in the detection of the DNA-hybridization event.

  19. Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH.

    Science.gov (United States)

    Ksiazczyk, T; Taciak, M; Zwierzykowski, Z

    2010-01-01

    This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploid Festuca pratensis and Lolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploid F. pratensis × L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) with L. perenne genomic DNA as a probe, and F. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. In F. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standard F. pratensis karyotypes. Losses of 45S rDNA loci were more frequent in L. perenne cultivars and intergeneric hybrids. Comparison of the F. pratensis and L. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location in L. perenne. A greater instability of F. pratensis-genome-like and L. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 in F. pratensis and on chromosome 3 in L. perenne are useful markers for these chromosomes in intergeneric Festuca × Lolium hybrids.

  20. Cryptic introgression of Dasypyrum villosum parental DNA in wheat lines derived from intergeneric hybridization.

    Science.gov (United States)

    Caceres, M E; Pupilli, F; Ceccarelli, M; Vaccino, P; Sarri, V; De Pace, C; Cionini, P G

    2012-01-01

    Cytogenetic and DNA molecular analyses have been carried out in 3 wheat introgression lines (ILs; CS×V58, CS×V59, and CS×V60) derived from Triticum aestivum cv. 'Chinese Spring' (CS) × Dasypyrum villosum(Dv) intergeneric hybridization. All lines, which showed several phenotypic differences compared to CS, had the same chromosome number (2n = 42) and structure as CS, and neither chromosomes nor chromatin from Dv were apparently added to their complement. However, Feulgen/DNA cytophotometry showed that there was more nuclear DNA in the lines than in the parental wheat (by 1.85%, 2.76%, and 1.26% in CS×V58, CS×V59, and CS×V60, respectively). Molecular investigation indicated the presence of Dv DNA in the ILs. AFLP analysis of genomic DNA from the ILs, CS, and Dv detected a total of 120 polymorphic bands, of which 7 (5.8%) were present in some or all the ILs and Dv but were absent in CS. PCR amplification, sequence analysis of amplicons, and Southern blot hybridization confirmed the presence of Dv-specific sequences in each of the ILs. These results indicate cryptic introgression of Dv DNA sequences into the genome of the ILs. Some implications of this finding are discussed. Copyright © 2011 S. Karger AG, Basel.

  1. Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1.

    Science.gov (United States)

    Bento, Joana C; Lane, Kristin D; Read, Erik K; Cerca, Nuno; Christie, Gail E

    2014-01-01

    The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.

  2. A hybrid swarm population of Pinus densiflora x P. sylvestris hybrids inferred from sequence analysis of chloroplast DNA and morphological characters

    Science.gov (United States)

    To confirm a hybrid swarm population of Pinus densiflora × P. sylvestris in Jilin, China and to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker (cpDNA SSR), needles and seeds from P. densiflora, P. syl...

  3. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    Science.gov (United States)

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.

  4. DNA Transfer from Wild Millet to Common Wheat by Asymmetric Somatic Hybridization

    Institute of Scientific and Technical Information of China (English)

    CHENGAi-Xia; XIAGuang-Min; CHENHui-Min

    2004-01-01

    Following differing periods of long-term subculture and selection, two types of calli of the same wheat ( Triticum aestivum L. cv. Jinan 177) were obtained. The one (named Cha 9) grew fast and easily formed cell suspensions that were non-regenerable, but the protoplasts possessed a high division capacity. The other (named 176) was regenerable, but the derived protoplasts grew slowly. Fusion combination between either Cha 9 or 176 protoplasts and UV-treated wild millet (SetaHa italica L. Beaur.) protoplasts failed to produce regenerated green plants. However, when the two types of wheat protoplasts were mixed together as recipient and fused with wild millet, green plants were obtained. The hybrid nature of regener-ated calli and plants was confirmed by the analysis of cytological, isozyme, 5S rDNA spacer sequences and random amplified polymorphic DNA (RAPD). The chloroplast genomes of hybrids were analyzed with several wheat-specific chloroplast microsatellite (simple sequence repeat, SSR) primers. A hybrid clone carrying recipient DNA of Cha 9 and 176, as well as both nuclear and chloroplast donor DNA had a high regeneration capacity and produced more vigorous green plants than did the other clones.

  5. Neighboring genes for DNA-binding proteins rescue male sterility in Drosophila hybrids.

    Science.gov (United States)

    Liénard, Marjorie A; Araripe, Luciana O; Hartl, Daniel L

    2016-07-19

    Crosses between closely related animal species often result in male hybrids that are sterile, and the molecular and functional basis of genetic factors for hybrid male sterility is of great interest. Here, we report a molecular and functional analysis of HMS1, a region of 9.2 kb in chromosome 3 of Drosophila mauritiana, which results in virtually complete hybrid male sterility when homozygous in the genetic background of sibling species Drosophila simulans. The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1 Both encode unrelated DNA-binding proteins, agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator. The contribution of each gene to hybrid male sterility was assessed by means of germ-line transformation, with constructs containing complete agt and Taf1 genomic sequences as well as various chimeric constructs. Both agt and Taf1 contribute about equally to HMS1 hybrid male sterility. Transgenes containing either locus rescue sterility in about one-half of the males, and among fertile males the number of offspring is in the normal range. This finding suggests compensatory proliferation of the rescued, nondysfunctional germ cells. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1 Our results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation.

  6. Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes

    Science.gov (United States)

    Kitajima, Akira; Nonaka, Keisuke; Yoshioka, Terutaka; Ohta, Satoshi; Goto, Shingo; Toyoda, Atsushi; Fujiyama, Asao; Mochizuki, Takako; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

    2016-01-01

    Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy–Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies. PMID:27902727

  7. Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes.

    Science.gov (United States)

    Shimizu, Tokurou; Kitajima, Akira; Nonaka, Keisuke; Yoshioka, Terutaka; Ohta, Satoshi; Goto, Shingo; Toyoda, Atsushi; Fujiyama, Asao; Mochizuki, Takako; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

    2016-01-01

    Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

  8. Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    Wen-Hui WEI; Wan-Peng ZHAO; Li-Jun WANG; Bo CHEN; Yun-Chang LI; Yun-Chun SONG

    2005-01-01

    In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t- 1 DNA was extracted from its genomic DNA, labeled with biotin- 11-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization (FISH)signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both C0t-1 DNA FISH banding patterns and chromosome morphology.

  9. A novel gold nanoparticle-DNA aptamer-based plasmonic chip for rapid and sensitive detection of bacterial pathogens

    DEFF Research Database (Denmark)

    Sun, Yi; Phuoc Long, Truong; Wolff, Anders

    2016-01-01

    Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers to be detac......Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers...... to be detached from AuNPs when interacting with bacteria. The new strategy greatly increases the sensitivity and specificity of chip-based whole-cell biosensing....

  10. Designing a Single-Molecule Biophysics Tool for Characterising DNA Damage for Techniques that Kill Infectious Pathogens Through DNA Damage Effects.

    Science.gov (United States)

    Miller, Helen; Wollman, Adam J M; Leake, Mark C

    2016-01-01

    Antibiotics such as the quinolones and fluoroquinolones kill bacterial pathogens ultimately through DNA damage. They target the essential type IIA topoisomerases in bacteria by stabilising the normally transient double-strand break state which is created to modify the supercoiling state of the DNA. Here we discuss the development of these antibiotics and their method of action. Existing methods for DNA damage visualisation, such as the comet assay and immunofluorescence imaging can often only be analysed qualitatively and this analysis is subjective. We describe a putative single-molecule fluorescence technique for quantifying DNA damage via the total fluorescence intensity of a DNA origami tile fully saturated with an intercalating dye, along with the optical requirements for how to implement these into a light microscopy imaging system capable of single-molecule millisecond timescale imaging. This system promises significant improvements in reproducibility of the quantification of DNA damage over traditional techniques.

  11. SWNT-DNA and SWNT-polyC hybrids: AFM study and computer modeling.

    Science.gov (United States)

    Karachevtsev, M V; Lytvyn, O S; Stepanian, S G; Leontiev, V S; Adamowicz, L; Karachevtsev, V A

    2008-03-01

    Hybrids of carbon single-walled nanotubes (SWNT) with fragmented single or double-stranded DNA (fss- or fds-DNA) or polyC were studied by Atom Force Microscopy (AFM) and computer modeling. It was found that fragments of the polymer wrap in several layers around the nanotube, forming a strand-like spindle. In contrast to the fss-DNA, the fds-DNA also forms compact structures near the tube surface due to the formation of self-assembly structures consisting of a few DNA fragments. The hybrids of SWNT with wrapped single-, double- or triple strands of the biopolymer were simulated, and it was shown that such structures are stable. To explain the reason of multi-layer polymeric coating of the nanotube surface, the energy of the intermolecular interactions between different components of polyC was calculated at the MP2/6-31++G** level as well as the interaction energy in the SWNT-cytosine complex.

  12. Combined RNA/DNA fluorescence in situ hybridization on whole-mount Drosophila ovaries.

    Science.gov (United States)

    Shpiz, Sergey; Lavrov, Sergey; Kalmykova, Alla

    2014-01-01

    DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity.

  13. Blue shift of CdSe/ZnS nanocrystal-labels upon DNA-hybridization

    Directory of Open Access Journals (Sweden)

    Palme Klaus

    2008-05-01

    Full Text Available Abstract Luminescence color multiplexing is one of the most intriguing benefits, which might occur by using semiconductor Quantum Dots (QDs as labels for biomolecules. It was found, that the luminescence of QDs can be quenched, and replaced by a luminescence peak at approximately 460 nm on hybridization with certain regions of Arabidopsis thaliana tissue. This effect is site selective, and it is unclear whether it occurs due to an energy transfer process, or due to quenching and scattering of the excitation light. The article describes methods for phase-transfer of differently coloured, hydrophobically ligated QDs, coupling of DNA strands to the QD's surface, and hybridization of the labelled DNA to different cell types of Arabidopsis thaliana. The reason for the luminescence blue-shift was studied systematically, and narrowed down to the above mentioned causes.

  14. Mitochondrial DNA sequence of the hybrid of Squaliobarbus curriculus (♀) × Ctenopharyngodon idella (♂).

    Science.gov (United States)

    Liu, Qiao-Lin; Liu, Min; Xiao, Tiao-Yi; Xu, Bao-Hong; Su, Jian-Ming

    2013-08-01

    In this work, we reported the complete mitochondrial DNA sequence of the hybrid of Squaliobarbus curriculus (♀) × Ctenopharyngodon idella (♂), which was obtained by artificial hybridization. The total length of the mitochondrial genome is 16,616 bp, with the base composition of 31.15% A, 25.02% T, 27.66% C, and 16.17% G. It contains 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and a major non-coding control region (D-loop region). The arrangement of these genes is the same as that found in the teleosts. All the protein initiation codons are ATG, except for COX1 that begins with GTG. The complete mitogenome of the hybrid of S. curriculus (♀) × C. idella (♂) provides an important data set for the study in genetic mechanism.

  15. Rapid high-throughput genotyping of HBV DNA using a modified hybridization-extension technique.

    Science.gov (United States)

    Bao, Han; Zhao, Wenliang; Ruan, Banjun; Wang, Qing; Zhao, Jinrong; Lei, Xiaoying; Wang, Weihua; Liu, Yonglan; Sun, Jianbing; Xiang, An; Guo, Yanhai; Yan, Zhen

    2013-11-07

    China has the highest incidence of hepatitis B virus (HBV) infection worldwide. HBV genotypes have variable impacts on disease pathogenesis and drug tolerance. We have developed a technically simple and accurate method for HBV genotyping that will be applicable to pre-treatment diagnosis and individualized treatment. Multiple sequence alignments of HBV genomes from GenBank were used to design primers and probes for genotyping of HBV A through H. The hybridization was carried out on nitrocellulose (NC) membranes with probes fixed in an array format, which was followed by hybrid amplification by an extension step with DNA polymerase to reinforce the double-stranded DNA hybrids on the NC membrane and subsequent visualization using an avidin-biotin system. Genotyping results were confirmed by DNA sequencing and bioinformatics analysis using the National Center for Biotechnology Information genotyping database, and compared with results from the line probe assay. The data show that multiple sequence alignment defined a 630 bp region in the HBV PreS and S regions that was suitable for genotyping. All genotyping significant single nucleotides in the region were defined. Two-hundred-and-ninety-one HBV-positive serum samples from Northwest Chinese patients were genotyped, and the genotyping rate from the new modified hybridization-extension method was 100% compared with direct sequencing. Compared with line probe assay, the newly developed method is superior, featuring reduced reaction time, lower risk of contamination, and increased accuracy for detecting single nucleotide mutation. In conclusion, a novel hybridization-extension method for HBV genotyping was established, which represents a new tool for accurate and rapid SNP detection that will benefit clinical testing.

  16. The constant region contributes to the antigenic specificity and renal pathogenicity of murine anti-DNA antibodies.

    Science.gov (United States)

    Xia, Yumin; Pawar, Rahul D; Nakouzi, Antonio S; Herlitz, Leal; Broder, Anna; Liu, Kui; Goilav, Beatrice; Fan, Manxia; Wang, Ling; Li, Quan-Zhen; Casadevall, Arturo; Putterman, Chaim

    2012-12-01

    Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9-11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9-11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9-11 hybridomas. We found that PL9-11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3>IgG2a>IgG1>IgG2b>IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9-11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.

  17. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  18. Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

    Science.gov (United States)

    Fichte, Manuela A H; Weyel, Xenia M M; Junek, Stephan; Schäfer, Florian; Herbivo, Cyril; Goeldner, Maurice; Specht, Alexandre; Wachtveitl, Josef; Heckel, Alexander

    2016-07-25

    We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

  19. Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea

    DEFF Research Database (Denmark)

    Jonach, Beata Renata; Boye, Mette; Stockmarr, Anders

    2014-01-01

    pathogens. The microorganisms that for decades have been associated with enteritis and diarrhea in suckling piglets are: rotavirus A, coronavirus, enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, Cryptosporidium spp., Giardia spp., Cystoisospora suis and Strongyloides ransomi...

  20. Method for isolating chromosomal DNA in preparation for hybridization in suspension

    Science.gov (United States)

    Lucas, Joe N.

    2000-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

  1. Use of conventional taxonomy, electrophoretic karyotyping and DNA-DNA hybridization for the classification of fermentative apiculate yeasts.

    Science.gov (United States)

    Vaughan-Martini, A; Angelini, P; Cardinali, G

    2000-07-01

    A taxonomic study was conducted that considered strains of the genera Hanseniaspora/Kloeckera held in the Industrial Yeasts Collection (DBVPG) of the Dipartimento di Biologia Vegetale of the Università di Perugia, Italy. Standard phenotypic as well as molecular criteria were considered in a effort to revisit the classification of these strains, some of which have been in the collection for about 50 years. Results of salient physiological tests showed that some of the DBVPG and type strains could not be identified by current taxonomic keys. Electrophoretic karyotypes were identical for some species, with the type strains of the seven accepted species showing only five distinct chromosomal patterns. DNA-DNA hybridization analyses, using a non-radioactive dot-blot technique, allowed for the distinction of taxa. The taxonomic implications of these results are discussed.

  2. Simultaneous in situ hybridization for DNA and RNA reveals the presence of HPV in the majority of cervical cancer cells.

    Science.gov (United States)

    D'Amato, L; Pilotti, S; Longoni, A; Donghi, R; Rilke, F

    1992-02-01

    Thirteen cases of invasive squamous cell carcinoma of the uterine cervix containing HPV types 16 or 18 DNA sequences, as detected by Southern blot analysis, were investigated by in situ hybridization on routine paraffin sections, using 35S nick-translated DNA probes. Simultaneous in situ hybridization for DNA and RNA showed that in ten out of 13 cases (77%) the percentage of tumor cells containing HPV 16 or 18 varied from 75 to 100%. In one case, harboring both in situ and invasive carcinoma, the same type of HPV DNA was detected in both components. This finding suggests that neoplastic cells retained the viral genome during progression to invasiveness.

  3. One-chip electronic detection of DNA hybridization using precision impedance-based CMOS array sensor.

    Science.gov (United States)

    Lee, Kang-Ho; Lee, Jeong-Oen; Sohn, Mi-Jin; Lee, Byunghun; Choi, Suk-Hwan; Kim, Sang Kyu; Yoon, Jun-Bo; Cho, Gyu-Hyeong

    2010-12-15

    This paper describes a label-free and fully electronic detection method of DNA hybridization, which is achieved through the use of a 16×8 microarray sensor in conjunction with a new type of impedance spectroscopy constructed with standard complementary metal-oxide-semiconductor (CMOS) technology. The impedance-based method is based on changes in the reactive capacitance and the charge-transfer resistance after hybridization with complementary DNA targets. In previously published label-free techniques, the measured capacitance presented unstable capacitive properties due to the parallel resistance that is not infinite and can cause a leakage by discharging the charge on the capacitor. This paper presents an impedance extraction method that uses excitation by triangular wave voltage, which enables a reliable measurement of both C and R producing a highly sensitive sensor with a stable operation independent of external variables. The system was fabricated in an industrial 0.35-μm 4-metal 2-poly CMOS process, integrating working electrodes and readout electronics into one chip. The integrated readout, which uses a parasitic insensitive integrator, achieves an enlarged detection range and improved noise performance. The maximum average relative variations of C and R are 31.5% and 68.6%, respectively, after hybridization with a 1 μM target DNA. The proposed sensor allows quantitative evaluation of the molecule densities on the chip with distinguishable variation in the impedance. This fully electronic microsystem has great potential for use with bioanalytical tools and point-of-care diagnosis.

  4. Bio-bar-code functionalized magnetic nanoparticle label for ultrasensitive flow injection chemiluminescence detection of DNA hybridization.

    Science.gov (United States)

    Bi, Sai; Zhou, Hong; Zhang, Shusheng

    2009-10-07

    A signal amplification strategy based on bio-bar-code functionalized magnetic nanoparticles as labels holds promise to improve the sensitivity and detection limit of the detection of DNA hybridization and single-nucleotide polymorphisms by flow injection chemiluminescence assays.

  5. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    Science.gov (United States)

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection.

  6. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were...

  7. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

    Science.gov (United States)

    Bünemann, H

    1982-11-25

    The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support. This correlation shows a characteristic course for a particular coupling method. DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage. Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports. Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).

  8. Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

    Science.gov (United States)

    Lau, Han Yih; Wu, Haoqi; Wee, Eugene J. H.; Trau, Matt; Wang, Yuling; Botella, Jose R.

    2017-01-01

    Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

  9. Titantium Dioxide Nanoparticles Assembled by DNA Molecules Hybridization and Loading of DNA Interacting Proteins.

    Science.gov (United States)

    Wu, Aiguo; Paunesku, Tatjana; Brown, Eric M B; Babbo, Angela; Cruz, Cecille; Aslam, Mohamed; Dravid, Vinayak; Woloschak, Gayle E

    2008-02-01

    This work demonstrates the assembly of TiO(2) nanoparticles with attached DNA oligonucleotides into a 3D mesh structure by allowing base pairing between oligonucleotides. A change of the ratio of DNA oligonucleotide molecules and TiO(2) nanoparticles regulates the size of the mesh as characterized by UV-visible light spectra, transmission electron microscopy and atomic force microscopy images. This type of 3D mesh, based on TiO(2)-DNA oligonucleotide nanoconjugates, can be used for studies of nanoparticle assemblies in material science, energy science related to dye-sensitized solar cells, environmental science as well as characterization of DNA interacting proteins in the field of molecular biology. As an example of one such assembly, proliferating cell nuclear antigen protein (PCNA) was cloned, its activity verified, and the protein was purified, loaded onto double strand DNA oligonucleotide-TiO(2) nanoconjugates, and imaged by atomic force microscopy. This type of approach may be used to sample and perhaps quantify and/or extract specific cellular proteins from complex cellular protein mixtures affinity based on their affinity for chosen DNA segments assembled into the 3D matrix.

  10. Chromosomal assignment of human DNA fingerprint sequences by simultaneous hybridization to arbitrarily primed PCR products from human/rodent monochromosome cell hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Jun; Sekiya, Takao [National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Navarro, J.M. [Burnham Institute, La Jolla, CA (United States)] [and others

    1996-05-15

    We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylong membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose. 23 refs., 3 figs., 2 tabs.

  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. Cytonuclear conflict in interpopulation hybrids: the role of RNA polymerase in mtDNA transcription and replication.

    Science.gov (United States)

    Ellison, C K; Burton, R S

    2010-03-01

    Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome.

  13. 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.

  14. rDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

    Science.gov (United States)

    Long, Hong; Chen, Chunli; Wang, Bing; Feng, Yanni

    2015-01-01

    The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins.

  15. Close sequence identity between ribosomal DNA episomes of the non-pathogenic Entamoeba dispar and pathogenic Entamoeba histolytica

    Indian Academy of Sciences (India)

    Jaishree Paul; Alok Bhattacharya; Sudha Bhattacharya

    2002-11-01

    Entamoeba dispar and Entamoeba histolytica are now recognized as two distinct species – the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes in E. histolytica. Here we report the analysis of ribosomal RNA genes in E. dispar. The rRNA genes of E. dispar, like their counterpart in E. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size of E. dispar rDNA circle was estimated to be 24.4 kb. The size was also confirmed by linearizing the circle with BsaHI, and by limited DNAseI digestion. The restriction map of the E. dispar rDNA circle showed close similarity to EhR1, the rDNA circle of E. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present in E. dispar. Partial sequencing of the cloned fragments of E. dispar rDNA and comparison with EhR1 revealed only 2.6% to 3.8% sequence divergence in the IGS. The region Tr and the adjoining PvuI repeats in the IGS of EhR1, which are missing in those E. histolytica strains that have one rDNA unit per circle, were present in the E. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1.6% in the two species. The most divergent sequence between E. histolytica and E. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.

  16. Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation.

    Science.gov (United States)

    López-Garrido, Javier; Casadesús, Josep

    2010-03-01

    DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.

  17. Nanofabrication Yields. Hybridization and Click-Fixation of Polycyclic DNA Nanoassemblies

    KAUST Repository

    Lundberg, Erik P.

    2011-09-27

    We demonstrate the stepwise assembly of a fully addressable polycyclic DNA hexagon nanonetwork for the preparation of a four-ring system, one of the biggest networks yet constructed from tripodal building blocks. We find that the yield exhibits a distinct upper level <100%, a fundamental problem of thermodynamic DNA assembly that appears to have been overlooked in the DNA nanotechnology literature. A simplistic model based on a single step-yield parameter y can quantitatively describe the total yield of DNA assemblies in one-pot reactions as Y = yduplex n, with n the number of hybridization steps. Experimental errors introducing deviations from perfect stoichiometry and the thermodynamics of hybridization equilibria contribute to decreasing the value of yduplex (on average y = 0.96 for our 10 base pair hybridization). For the four-ring system (n = 31), the total yield is thus less than 30%, which is clearly unsatisfactory if bigger nanoconstructs of this class are to be designed. Therefore, we introduced site-specific click chemistry for making and purifying robust building blocks for future modular constructs of larger assemblies. Although the present yield of this robust module was only about 10%, it demonstrates a first step toward a general fabrication approach. Interestingly, we find that the click yields follow quantitatively a binomial distribution, the predictability of which indicates the usefulness of preparing pools of pure and robust building blocks in this way. The binomial behavior indicates that there is no interference between the six simultaneous click reactions but that step-yield limiting factors such as topological constraints and Cu(I) catalyst concentration are local and independent. © 2011 American Chemical Society.

  18. DNA aptamer release from the DNA-SWNT hybrid by protein recognition.

    Science.gov (United States)

    Yoo, Chang-Hyuk; Jung, Seungwon; Bae, Jaehyun; Kim, Gunn; Ihm, Jisoon; Lee, Junghoon

    2016-02-14

    Here we show the formation of the complex between a DNA aptamer and a single-walled carbon nanotube (SWNT) and its reaction with its target protein. The aptamer, which is specifically bound with thrombin, the target protein in this study, easily wraps and disperses the SWNT by noncovalent π-π stacking.

  19. DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948.

    Science.gov (United States)

    Labeda, D P; Lyons, A J

    1992-02-01

    DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).

  20. Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in Pelargonium.

    Science.gov (United States)

    Weihe, Andreas; Apitz, Janina; Pohlheim, Frank; Salinas-Hartwig, Annabel; Börner, Thomas

    2009-12-01

    Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330-351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargonium zonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes.

  1. Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

    Science.gov (United States)

    Umemura, Kazuo; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

    2016-12-01

    Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA-SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA-SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA-SWNT hybrids. The morphology of the SSB-ssDNA-SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB-ssDNA-SWNT hybrids showed much larger variance than the ssDNA-SWNT hybrids.

  2. Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Au(nano)-CNT/PAN(nano) films.

    Science.gov (United States)

    Zhou, Na; Yang, Tao; Jiang, Chen; Du, Meng; Jiao, Kui

    2009-01-15

    A polyaniline nanofibers (PAN(nano))/carbon paste electrode (CPE) was prepared via dopping PAN(nano) in the carbon paste. The nanogold (Au(nano)) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN(nano)/CPE. The immobilization and hybridization of the DNA probe on the Au(nano)-CNT/PAN(nano) films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)(6)](3-/4-) as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R(et)) of the electrode surface increased after the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films and rose further after the hybridization of the probe DNA. The remarkable difference between the R(et) value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au(nano)-CNT/PAN(nano) films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 x 10(-12)mol/L to 1.0 x 10(-6)mol/L with a detection limit of 5.6 x 10(-13)mol/L.

  3. 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.

  4. HMGA1a protein unfolds or refolds synthetic DNA-chromophore hybrid polymers: a chaperone-like behavior.

    Science.gov (United States)

    Wan, Wei; Wang, Wei; Li, Alexander D Q

    2008-01-25

    High group mobility protein, HMGA1a, was found to play a chaperone-like role in the folding or unfolding of hybrid polymers that contained well-defined synthetic chromophores and DNA sequences. The synthetic and biological hybrid polymers folded into hydrophobic chromophoric nanostructures in water, but existed as partially unfolded configurations in pH or salt buffers. The presence of HMGA1a induced unfolding of the hybrid DNA-chromophore polymer in pure water, whereas the protein promoted refolding of the same polymer in various pH or salt buffers. The origin of the chaperone-like properties probably comes from the ability of HMGA1a to reversibly bind both synthetic chromophores and single stranded DNA. The unfolding mechanisms and the binding stoichiometry of protein-hybrid polymers depended on the sequence of the synthetic polymers.

  5. DNA hybridization-induced reorientation of liquid crystal anchoring at the nematic liquid crystal/aqueous interface.

    Science.gov (United States)

    Price, Andrew D; Schwartz, Daniel K

    2008-07-01

    Interactions between DNA and an adsorbed cationic surfactant at the nematic liquid crystal (LC)/aqueous interface were investigated using polarized and fluorescence microscopy. The adsorption of octadecyltrimethylammonium bromide (OTAB) surfactant to the LC/aqueous interface resulted in homeotropic (untilted) LC alignment. Subsequent adsorption of single-stranded DNA (ssDNA) to the surfactant-laden interface modified the interfacial structure, resulting in a reorientation of the LC from homeotropic alignment to an intermediate tilt angle. Exposure of the ssDNA/OTAB interfacial complex to its ssDNA complement induced a second change in the interfacial structure characterized by the nucleation, growth, and coalescence of lateral regions that induced homeotropic LC alignment. Fluorescence microscopy showed explicitly that the complement was colocalized in the same regions as the homeotropic domains. Exposure to noncomplementary ssDNA caused no such response, suggesting that the homeotropic regions were due to DNA hybridization. This hybridization occurred in the vicinity of the interface despite the fact that the conditions in bulk solution were such that hybridization did not occur (high stringency), suggesting that the presence of the cationic surfactant neutralized electrostatic repulsion and allowed for hydrogen bonding between DNA complements. This system has potential for label-less and portable DNA detection. Indeed, LC response to ssDNA target was detected with a lower limit of approximately 50 fmol of complement and was sufficiently selective to differentiate a one-base-pair mismatch in a 16-mer target.

  6. 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.

  7. Characterization of the structural and protein recognition properties of hybrid PNA-DNA four-way junctions.

    Science.gov (United States)

    Iverson, Douglas; Serrano, Crystal; Brahan, Ann Marie; Shams, Arik; Totsingan, Filbert; Bell, Anthony J

    2015-12-01

    The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg(+2). For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg(+2). The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.

  8. Monitoring DNA hybridization and thermal dissociation at the silica/water interface using resonantly enhanced second harmonic generation spectroscopy.

    Science.gov (United States)

    Azam, Md Shafiul; Gibbs-Davis, Julianne M

    2013-09-03

    The immobilization of oligonucleotide sequences onto glass supports is central to the field of biodiagnostics and molecular biology with the widespread use of DNA microarrays. However, the influence of confinement on the behavior of DNA immobilized on silica is not well understood owing to the difficulties associated with monitoring this buried interface. Second harmonic generation (SHG) is an inherently surface specific technique making it well suited to observe processes at insulator interfaces like silica. Using a universal 3-nitropyrolle nucleotide as an SHG-active label, we monitored the hybridization rate and thermal dissociation of a 15-mer of DNA immobilized at the silica/aqueous interface. The immobilized DNA exhibits hybridization rates on the minute time scale, which is much slower than hybridization kinetics in solution but on par with hybridization behavior observed at electrochemical interfaces. In contrast, the thermal dissociation temperature of the DNA immobilized on silica is on average 12 °C lower than the analogous duplex in solution, which is more significant than that observed on other surfaces like gold. We attribute the destabilizing affect of silica to its negatively charged surface at neutral pH that repels the hybridizing complementary DNA.

  9. Photopolymerization of polydiacetylene in hybrid liposomes: effect of polymerization on stability and response to pathogenic bacterial toxins.

    Science.gov (United States)

    Thet, Naing Tun; Jamieson, William David; Laabei, Maisem; Mercer-Chalmers, June D; Jenkins, A Toby A

    2014-05-22

    Liposomes containing lipids and polydiacetylene (PDA) are hybrid systems encompassing both a fluid phospholipid membrane and a polymer scaffold (PDA). However, the biophysical role of PDA in such liposomes is not well understood. In this report, we studied the effects of photopolymerization of PDA on the stability of lipid-PDA liposomes, and their sensitivity to selected purified toxins and bacterial supernatants, using a fluorescence assay. Of the three different types of liposomes with variable lipid chain lengths that were chosen, the degree of polymerization had a significant impact on the long-term stability, and response, to external microbial exotoxins secreted by pathogenic bacteria, namely, Staphylococcus aureus and Pseudomonas aeruginosa. The degree of polymerization of TCDA played an important role in lipid-chain-length-dependent stabilization of lipid-PDA liposomes, as well as in their response to bacterial toxins of S. aureus and P. aeruginosa.

  10. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  11. Bright luminescence from pure DNA-curcumin–based phosphors for bio hybrid light-emitting diodes

    Science.gov (United States)

    Reddy, M. Siva Pratap; Park, Chinho

    2016-08-01

    Recently, significant advances have occurred in the development of phosphors for bio hybrid light-emitting diodes (Bio-HLEDs), which have created brighter, metal-free, rare-earth phosphor-free, eco-friendly, and cost-competitive features for visible light emission. Here, we demonstrate an original approach using bioinspired phosphors in Bio-HLEDs based on natural deoxyribonucleic acid (DNA)-curcumin complexes with cetyltrimethylammonium (CTMA) in bio-crystalline form. The curcumin chromophore was bound to the DNA double helix structure as observed using field emission tunnelling electron microscopy (FE-TEM). Efficient luminescence occurred due to tightly bound curcumin chromophore to DNA duplex. Bio-HLED shows low luminous drop rate of 0.0551 s‑1. Moreover, the solid bio-crystals confined the activating bright luminescence with a quantum yield of 62%, thereby overcoming aggregation-induced quenching effect. The results of this study herald the development of commercially viable large-scale hybrid light applications that are environmentally benign.

  12. 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.

  13. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    Science.gov (United States)

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis.

  14. Thermodynamics for the Formation of Double-Stranded DNA-Single-Walled Carbon Nanotube Hybrids.

    Science.gov (United States)

    Shiraki, Tomohiro; Tsuzuki, Akiko; Toshimitsu, Fumiyuki; Nakashima, Naotoshi

    2016-03-24

    For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA.

  15. Phylogenetic relationships and natural hybridization in rabbitfishes (Teleostei: Siganidae) inferred from mitochondrial and nuclear DNA analyses.

    Science.gov (United States)

    Kuriiwa, Kaoru; Hanzawa, Naoto; Yoshino, Tetsuo; Kimura, Seishi; Nishida, Mutsumi

    2007-10-01

    Phylogenetic relationships of rabbitfishes (the family Siganidae), ecologically important components as primary consumers in coral reef communities, were studied using mitochondrial cytochrome b gene and nuclear ITS1 (internal transcribed spacer 1) sequence analyses. The analyses of 19 out of 22 species known in the Western Pacific region revealed that siganids are genetically clustered into three major clades, which are characterized by some morphological and ecological traits. Between closely related species, such as Siganus guttatus-S. lineatus and S. virgatus-S. doliatus, and also between two morphs recognized in S. corallinus, small but discernible genetic differentiation was found, implying that the components of each pair are incipient species. On the other hand, between some species, such as S. fuscescens-S. canaliculatus and S. unimaculatus-S.vulpinus, individuals of the components of each pair were found to construct a genetic mosaic, suggesting that the components are genetic color morphs within a single biological species, respectively. Moreover, evidence from morphological characters, mtDNA, and nuclear DNA gave an inconsistent picture of identity and relationships for several individuals. They were regarded as hybrids or individuals with hybrid origin. Such instances were observed not only between closely related species, such as S. guttatus-S. lineatus, S. virgatus-S. doliatus, and two morphs (incipient species) in S. corallinus, respectively, but also between distantly related ones, such as S. corallinus-S. puellus. In fact, more than half of the species examined (11/20, when treating the two morphs in S. corallinus as independent species) were involved in hybridization. These suggest that hybridization is much more prevalent in marine fishes than previously assumed, and may have some relevance to their diversification.

  16. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Science.gov (United States)

    Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

  17. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Directory of Open Access Journals (Sweden)

    Andrea J Adams

    Full Text Available Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM to Macherey-Nagel DNA FFPE (MN, test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90% when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections, current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from

  18. 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.

  19. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

    Directory of Open Access Journals (Sweden)

    Dolores L. Guzmán-Herrador

    2017-08-01

    Full Text Available We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

  20. [Mitochondrial DNA polymorphisms shared between modern humans and neanderthals: adaptive convergence or evidence for interspecific hybridization?].

    Science.gov (United States)

    Maliarchuk, B A

    2013-09-01

    An analysis of the variability of the nucleotide sequences in the mitochondrial genome of modern humans, neanderthals, Denisovans, and other primates has shown that there are shared polymorphisms at positions 2758 and 7146 between modern Homo sapiens (in phylogenetic cluster L2'3'4'5'6) and Homo neanderthalensis (in the group of European neanderthals younger than 48000 years). It is suggested that the convergence may be due to adaptive changes in the mitochondrial genomes of modern humans and neanderthals or interspecific hybridization associated with mtDNA recombination.

  1. Macroscopic assembly by optical control of zmol-level DNA hybridization

    Science.gov (United States)

    Iida, Takuya; Nishimura, Yushi; Tamura, Mamoru; Nishida, Keisuke; Ito, Syoji; Tokonami, Shiho

    2017-04-01

    Remote acceleration of a molecular recognition will open an avenue for the control of various biological functions. Here, we have developed a new principle for the rapid macroscopic assembly based on the light-induced molecular recognition via nanoparticles. Remarkably, as an application of this principle, we have demonstrated the submillimetre network formation triggered by light-induced hybridization of zmol-level DNA within a few minutes. This finding will be used for the rapid and highly sensitive genetic screening without fluorescent labeling.

  2. 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...

  3. Physical mapping of 18S-25S rDNA and 5S rDNA in Lupinus via fluorescent in situ hybridization.

    Science.gov (United States)

    Naganowska, Barbara; Zielińska, Anna

    2002-01-01

    Double-target fluorescent in situ hybridization (FISH) was used to determine the genomic distribution of ribosomal RNA genes in five Lupinus species: L. cosentinii (2n=32), L. pilosus (2n=42), L. angustifolius (2n=40), L. luteus (2n=52) and L. mutabilis (2n=48). 18S-25S rDNA and 5S rDNA were used as probes. Some interspecific variation was observed in the number and size of the 18S-25S rDNA loci. All the studied species had one chromosome pair carrying 5S rDNA.

  4. Bioinspired Synthesis of All-in-One Organic-Inorganic Hybrid Nanoflowers Combined with a Handheld pH Meter for On-Site Detection of Food Pathogen.

    Science.gov (United States)

    Ye, Ranfeng; Zhu, Chengzhou; Song, Yang; Lu, Qian; Ge, Xiaoxiao; Yang, Xu; Zhu, Mei-Jun; Du, Dan; Li, He; Lin, Yuehe

    2016-06-01

    With a mild elaborately bioinspired one-pot process, Con A-GOx-CaHPO4 nanoflowers are prepared. Employing the as-prepared all-in-one hybrid nanoflowers as signal tags, a simple but potentially powerful amplification biosensing technology for the detection of food pathogen with excellent simplicity, portability, sensitivity, and adaptability is achieved.

  5. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  6. Limits of RNA 2'-OH Mimicry by Fluorine: Crystal Structure of Bacillus halodurans RNase H Bound to a 2'-FRNA:DNA Hybrid.

    Science.gov (United States)

    Pallan, Pradeep S; Prakash, Thazha P; de Leon, Arnie R; Egli, Martin

    2016-09-27

    RNase H1 cleaves the RNA strand of RNA:DNA hybrids. Replacement of RNA 2'-hydroxyls by fluorine (FRNA) is commonly used to stabilize aptamers and siRNAs. However, FRNA:DNA hybrids fail to elicit RNase H activity. The underlying reasons are unclear, as 2'-OH groups are not directly involved in cleavage. We determined the crystal structure of Bacillus halodurans RNase H bound to a FRNA:DNA hybrid. The structure points to dynamic (slippage of the FRNA:DNA hybrid relative to the enzyme), geometric (different curvatures of FRNA:DNA and RNA:DNA hybrids), and electronic reasons (Mg(2+) absent from the active site of the FRNA:DNA complex) for the loss of RNaseH activity.

  7. Comparison of pathogen DNA isolation methods from large volumes of whole blood to improve molecular diagnosis of bloodstream infections.

    Directory of Open Access Journals (Sweden)

    Anne J M Loonen

    Full Text Available For patients suffering from bloodstream infections (BSI molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml, the novel non-enzymatic procedure (Polaris, 1-5 ml, and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl. These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67% and EasyMAG (58-79%. For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%. The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction vs. 2 hours (MolYsis. In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.

  8. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

    OpenAIRE

    Meier, A.; Persing, D. H.; Finken, M; Böttger, E C

    1993-01-01

    Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are...

  9. Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in Oreochromis mossambicus, O. urolepis hornorum and their hybrid

    Indian Academy of Sciences (India)

    Hua Ping Zhu; Mai Xin Lu; Feng Ying Gao; Zhang Han Huang; Li Ping Yang; Jain Fang Gui

    2010-08-01

    In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female × O. u. hornorum male. An identical karyotype (($2n = 44$, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.

  10. Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus.

    Science.gov (United States)

    Rech, Gabriel E; Sanz-Martín, José M; Anisimova, Maria; Sukno, Serenella A; Thon, Michael R

    2014-09-04

    Natural selection leaves imprints on DNA, offering the opportunity to identify functionally important regions of the genome. Identifying the genomic regions affected by natural selection within pathogens can aid in the pursuit of effective strategies to control diseases. In this study, we analyzed genome-wide patterns of selection acting on different classes of sequences in a worldwide sample of eight strains of the model plant-pathogenic fungus Colletotrichum graminicola. We found evidence of selective sweeps, balancing selection, and positive selection affecting both protein-coding and noncoding DNA of pathogenicity-related sequences. Genes encoding putative effector proteins and secondary metabolite biosynthetic enzymes show evidence of positive selection acting on the coding sequence, consistent with an Arms Race model of evolution. The 5' untranslated regions (UTRs) of genes coding for effector proteins and genes upregulated during infection show an excess of high-frequency polymorphisms likely the consequence of balancing selection and consistent with the Red Queen hypothesis of evolution acting on these putative regulatory sequences. Based on the findings of this work, we propose that even though adaptive substitutions on coding sequences are important for proteins that interact directly with the host, polymorphisms in the regulatory sequences may confer flexibility of gene expression in the virulence processes of this important plant pathogen. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Evidence for and Localization of Vegetative Viral DNA Replication by Autoradiographic Detection of RNA·DNA Hybrids in Sections of Tumors Induced by Shope Papilloma Virus

    Science.gov (United States)

    Orth, Gérard; Jeanteur, Philippe; Croissant, Odile

    1971-01-01

    The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis. Images PMID:4331563

  12. Chromosomal mapping of specific DNA gains and losses in solid tumors using comparative genomic hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Schrock, E.; Manoir, S. du; Speicher, M. [National Center for Human Genome Research, Bethesda, MD (United States)] [and others

    1994-09-01

    Comparative genomic hybridization (CGH) is a new molecular cytogenetic technique that is based on two color FISH and quantitative digital imaging microscopy. CGH is used to comprehensively survey tumor genomes for copy number changes and to determine the map position of amplification sites on normal reference chromosomes. CGH was used to analyze 107 different solid tumors, including 16 low grade astrocytomas, 15 recurrent astrocytic tumors, 13 high grade astrocytomas, 13 small cell lung cancers (SCLC), 14 breast cancer samples (7 diploid and 7 aneupoid tumors), 18 chromophobe renal cell carcinomas and 5 seminomas. Tumor DNA was extracted from frozen tissue, autopic material and formalin fixed, paraffin-embedded tissue samples. Our results revealed tumor specific gains and losses of certain chromosomes or chromosomal subregions (e.g., chromosomes 7 and 10 in glioblastomas, chromosomes 3 and 5 in SCLC). Numerous DNA-amplifications were mapped on reference metaphase and prometaphase chromosomes. The frequent amplification of the EGFR gene (malignant gliomas), protooncogenes of the myc family (SCLC) and of c-myc, int-2 and c-erbB2 (breast cancer) was confirmed. Many additional amplification sites, however, were mapped that were not described before. The results of CGH analysis were independently confirmed by means of cytogenetic banding analysis, interphase cytogenetics with region specific DNA-clones, Southern-Blot analysis, DNA-cytometry and studies of loss of heterozygosity.

  13. A G-Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

    Institute of Scientific and Technical Information of China (English)

    邵从英; 鲁娜; 孙登明

    2012-01-01

    A heroin-binding DNA G-quadruplex (also known as a heroin aptamer or DNAzyme) has been previously re- ported to be able to enhance the peroxidase activity of heroin. In this work, we described a DNAzyme structure that had an effector-recognizing part appearing as a single stranded DNA linkage flanked by two split G-quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G-quadruplex halves and thus efficiently suppressed the enzymatic activity of the G-quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re-engineered G-quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G-quadruplex is an especially useful module to design low-cost and label-free sensors toward various biologically or environmentally interesting targets.

  14. Locational diversity of alpha satellite DNA and intergeneric hybridization aspects in the Nomascus and Hylobates genera of small apes.

    Directory of Open Access Journals (Sweden)

    Sudarath Baicharoen

    Full Text Available Recently, we discovered that alpha satellite DNA has unique and genus-specific localizations on the chromosomes of small apes. This study describes the details of alpha satellite localization in the genera Nomascus and Hylobates and explores their usefulness in distinguishing parental genome sets in hybrids between these genera. Fluorescence in situ hybridization was used to establish diagnostic criteria of alpha satellite DNA markers in discriminating small ape genomes. In particular we established the genus specificity of alpha satellite distribution in three species of light-cheeked gibbons (Nomascus leucogenys, N. siki, and N. gabriellae in comparison to that of Hylobates lar. Then we determined the localization of alpha satellite DNA in a hybrid individual which resulted from a cross between these two genera. In Nomascus the alpha satellite DNA blocks were located at the centromere, telomere, and four interstitial regions. In Hylobates detectable amounts of alpha satellite DNA were seen only at centromeric regions. The differences in alpha satellite DNA locations between Nomascus and Hylobates allowed us to easily distinguish the parental chromosomal sets in the genome of intergeneric hybrid individuals found in Thai and Japanese zoos. Our study illustrates how molecular cytogenetic markers can serve as diagnostic tools to identify the origin of individuals. These molecular tools can aid zoos, captive breeding programs and conservation efforts in managing small apes species. Discovering more information on alpha satellite distribution is also an opportunity to examine phylogenetic and evolutionary questions that are still controversial in small apes.

  15. Locational diversity of alpha satellite DNA and intergeneric hybridization aspects in the Nomascus and Hylobates genera of small apes.

    Science.gov (United States)

    Baicharoen, Sudarath; Miyabe-Nishiwaki, Takako; Arsaithamkul, Visit; Hirai, Yuriko; Duangsa-ard, Kwanruen; Siriaroonrat, Boripat; Domae, Hiroshi; Srikulnath, Kornsorn; Koga, Akihiko; Hirai, Hirohisa

    2014-01-01

    Recently, we discovered that alpha satellite DNA has unique and genus-specific localizations on the chromosomes of small apes. This study describes the details of alpha satellite localization in the genera Nomascus and Hylobates and explores their usefulness in distinguishing parental genome sets in hybrids between these genera. Fluorescence in situ hybridization was used to establish diagnostic criteria of alpha satellite DNA markers in discriminating small ape genomes. In particular we established the genus specificity of alpha satellite distribution in three species of light-cheeked gibbons (Nomascus leucogenys, N. siki, and N. gabriellae) in comparison to that of Hylobates lar. Then we determined the localization of alpha satellite DNA in a hybrid individual which resulted from a cross between these two genera. In Nomascus the alpha satellite DNA blocks were located at the centromere, telomere, and four interstitial regions. In Hylobates detectable amounts of alpha satellite DNA were seen only at centromeric regions. The differences in alpha satellite DNA locations between Nomascus and Hylobates allowed us to easily distinguish the parental chromosomal sets in the genome of intergeneric hybrid individuals found in Thai and Japanese zoos. Our study illustrates how molecular cytogenetic markers can serve as diagnostic tools to identify the origin of individuals. These molecular tools can aid zoos, captive breeding programs and conservation efforts in managing small apes species. Discovering more information on alpha satellite distribution is also an opportunity to examine phylogenetic and evolutionary questions that are still controversial in small apes.

  16. Observation of microarray DNA hybridization using surface plasmon resonance phase-shift interferometry

    Science.gov (United States)

    Chen, Shean-Jen; Tsou, C.-Y.; Chen, Y.-K.; Su, Y.-T.

    2004-06-01

    Surface plasmon resonance phase-shift interferometry (SPR-PSI) is a novel technique which combines SPR and modified Mach-Zehnder phase-shifting interferometry to measure the spatial phase variation caused by biomolecular interactions upon a sensing chip. The SPR-PSI imaging system offers high resolution and high-throughout screening capabilities for microarray DNA hybridization without the need for additional labeling, and provides valuable real-time quantitative information. Current SPR-PSI imaging systems measure the spatial phase variation caused by tiny biomolecular changes on the sensing interface by means of a five-step phase reconstruction algorithm and a novel multichannel least mean squares (MLMS) phase unwrapping algorithm. The SPR-PSI imaging system has an enhanced detection limit of 2.5 × 10-7 refraction index change, a long-term phase stability of π/100 in 30 minutes, and a spatial phase resolution of π/500 with a lateral resolution of 10μm. This study successfully demonstrates the kinetic and label-free observation of 5-mer DNA microarray hybridization.

  17. Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination.

    Directory of Open Access Journals (Sweden)

    Chris R Smith

    Full Text Available BACKGROUND: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events. METHODOLOGY/PRINCIPAL FINDINGS: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP. Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination. CONCLUSIONS/SIGNIFICANCE: These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic

  18. UV-Induced DNA Damage Promotes Resistance to the Biotrophic Pathogen Hyaloperonospora parasitica in Arabidopsis1[C][OA

    Science.gov (United States)

    Kunz, Bernard A.; Dando, Paige K.; Grice, Desma M.; Mohr, Peter G.; Schenk, Peer M.; Cahill, David M.

    2008-01-01

    Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response. PMID:18667719

  19. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

    Science.gov (United States)

    Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

    2016-05-15

    Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.

    Science.gov (United States)

    He, Weiguo; Qin, Qinbo; Liu, Shaojun; Li, Tangluo; Wang, Jing; Xiao, Jun; Xie, Lihua; Zhang, Chun; Liu, Yun

    2012-01-01

    Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

  1. Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.

    Directory of Open Access Journals (Sweden)

    Weiguo He

    Full Text Available Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100 × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48 were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC and only class IV from their male parent (TC. Triploid hybrids inherited class II and class III from their female parent (RCC and class IV from their male parent (TC. Tetraploid hybrids gained class II and class III from their female parent (RCC, and generated a new 5S rDNA sequence (designated class I-N. The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

  2. Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

    Science.gov (United States)

    Unno, Hirotaka; Inada, Mika; Nakamura, Akiyoshi; Hashimoto, Michie; Ito, Keiko; Hashimoto, Koji; Nikaido, Masaru; Hayashi, Tomohito; Hata, Eiji; Katsuda, Ken; Kiku, Yoshio; Tagawa, Yuichi; Kawai, Kazuhiro

    2015-08-01

    A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

  3. A highly oriented hybrid microarray modified electrode fabricated by a template-free method for ultrasensitive electrochemical DNA recognition

    Science.gov (United States)

    Shi, Lei; Chu, Zhenyu; Dong, Xueliang; Jin, Wanqin; Dempsey, Eithne

    2013-10-01

    Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors.Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and

  4. DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

    Directory of Open Access Journals (Sweden)

    Wanshan Xiong

    Full Text Available DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

  5. Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability.

    Science.gov (United States)

    Wan, Lan; Chen, Qiaoshu; Liu, Jianbo; Yang, Xiaohai; Huang, Jin; Li, Li; Guo, Xi; Zhang, Jue; Wang, Kemin

    2016-04-11

    A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 μm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.

  6. Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector Borne Diseases: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Berlin L. Londono-Renteria

    2016-09-01

    Full Text Available Chronic exposure to antigens may favor the production of IgG4 antibodies over other antibody types. Recent studies have shown that up to a 30% of normal human IgG4 is bi-specific and is able to recognize two antigens of different nature. A requirement for this specificity is the presence of both eliciting antigens in the same time and at the same place where the immune response is induced. During transmission of most vector-borne diseases, the pathogen is delivered to the vertebrate host along with the arthropod saliva during blood feeding and previous studies have shown the existence of IgG4 antibodies against mosquito salivary allergens. However, there is very little ongoing research or information available regarding IgG4 bi-specificity with regards to infectious disease, particularly during immune responses to vector-borne diseases such as malaria, filariasis or dengue virus infection. Here, we provide background information and present our hypothesis that IgG4 may not only be a useful tool to measure exposure to infected mosquito bites, but that these bi-specific antibodies may also play an important role in modulation of the immune response against malaria and other vector-borne diseases in endemic settings.

  7. DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

    Science.gov (United States)

    Guo, Qi; Han, Jiao-Jiao; Shan, Shan; Liu, Dao-Feng; Wu, Song-Song; Xiong, Yong-Hua; Lai, Wei-Hua

    2016-12-15

    This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb-E. coli O157:H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.

  8. Hybridization chain reaction-based fluorescence immunoassay using DNA intercalating dye for signal readout.

    Science.gov (United States)

    Deng, Yan; Nie, Ji; Zhang, Xiao-hui; Zhao, Ming-Zhe; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2014-07-07

    A novel format of fluorescence immunosorbent assay based on the hybridization chain reaction (HCR) using a DNA intercalating dye for signal readout was constructed for the sensitive detection of targets, both in competitive and sandwich modes. In this platform, the capture and recognition processes are based on immunoreactions and the signal amplification depends on the enzyme-free, isothermal HCR-induced labelling event. After a competitive or a sandwich immunoreaction, a biotinylated capture DNA was bound to a biotinylated signal antibody through avidin, and triggered the HCR by two specific hairpins into a nicked double helix. Gene Finder (GF), a fluorescent probe for double-strand DNA, was intercalated in situ into the amplified chain to produce the fluorescence signal. The limit of detection (LOD) for rabbit IgG in competitive mode by HCR/GF immunoassay was improved at least 100-fold compared with the traditional fluorescence immunoassay using the fluorescein isothiocyanate-labelled-streptavidin or fluorescein isothiocyanate-labelled second antibody as the signal readout. The proposed fluorescence immunoassay was also demonstrated by using α-fetoprotein as the model target in sandwich mode, and showed a wide linear range from 28 ng mL(-1) to 20 μg mL(-1) with a LOD of 6.0 ng mL(-1). This method also showed satisfactory analysis in spiked human serum, which suggested that it might have great potential for versatile applications in life science and point-of-care diagnostics.

  9. Electrochemical sensing of DNA immobilization and hybridization based on carbon nanotubes/nano zinc oxide/chitosan composite film

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Tao Yang; Da Ming Huang; Kui Jiao

    2008-01-01

    A novel electrochemical DNA biosensor based on zinc oxide (ZnO) nanoparticles and multi-walled carbon nanotubes (MWNTs)for DNA immobilization and enhanced hybridization detection is presented. The MWNTs/nano ZnO/chitosan composite filmmodified glassy carbon electrode (MWNTs/ZnO/CHIT/GCE) was fabricated and DNA probes were immobilized on the electrodesurface. The hybridization events were monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as anindicator. The sensor can effectively discriminate different DNA sequences related to PAT gene in the.transgenic corn, with adetection limit of 2.8×10-12 mol/L of target sequence.2008 Kui Jiao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  10. A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR.

    Science.gov (United States)

    Li, M; Ishiguro, Y; Kageyama, K; Zhu, Z

    2015-08-01

    Most of the current research into the quantification of soil-borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co-extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real-time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample-to-sample basis and were DNA control for the normalization of DNA extraction by real-time PCR. By combining two different efficient soil DNA extraction methods, the developed quantification method dramatically improved the results. This study also proves that the developed normalization method is necessary and useful for the accurate quantification of soil-borne plant pathogenic oomycetes. © 2015 The Society for Applied Microbiology.

  11. Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

    Directory of Open Access Journals (Sweden)

    Ceren Sengiz

    2015-09-01

    Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

  12. Detection of Eleven Pathogenic Bacterium Using DNA Microarray Combined With Multiplex PCR%多重PCR结合基因芯片技术检测11种致病菌方法的建立

    Institute of Scientific and Technical Information of China (English)

    贺晨; 孙鸿燕; 邵丽筠; 刘金华; 张春秀; 刘娅

    2011-01-01

    Objective To establish a rapid ,accurate detection method for 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR .Methods The special genes of these Bacterium including Shigella, Listeria monocytogenes ,Escherichi-a coli O157, Staphylococcus aureus , Bacillus cereus , Campylobacter jejuni, Proteusbacillus vulgaris , C . perfringens , Vibiro para-haemolyticus ,Salmonella typhi ,Salmonella enteritidis were as target genes for multiplex PCR respectively ,then primers and captured oligonucleotide probes were designed and synthesized .After multiplex PCR system and hybridization reaction were optimized ,the multr-plex PCR products were hybridizes with DNA microarray ,which contained specific probes of eleven pathogenic bacterium .Scanner was used to determinant the types of bacterium .Results The DNA microarray assay can detect 11 kinds of pathogenic bacterium specially .The sensitivity of the DNA may reach 2?0-3 ng .Conclusion To detect 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR is specific ,sensiu've ,practical.%目的 建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法.方法 筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的 基因.设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片.将多重PCR扩增产物与带有11种特异探针的基因芯片杂交.用扫描仪扫描,判定细菌种类.结果 该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng.结论 多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性.

  13. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

    OpenAIRE

    1988-01-01

    We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

  14. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

    2013-02-19

    We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

  15. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Ortíz-Hernández, Brenda L.; Dávila-Rodríguez, Martha I.; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

    2013-01-01

    We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1. PMID:23429197

  16. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH

    Directory of Open Access Journals (Sweden)

    Jaime Gosálvez

    2013-02-01

    Full Text Available We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH. A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL, 10 with high-grade SIL (HG-SIL, and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

  17. Differential gene expression for Curvularia eragrostidis pathogenic incidence in crabgrass (Digitaria sanguinalis revealed by cDNA-AFLP analysis.

    Directory of Open Access Journals (Sweden)

    Jianshu Wang

    Full Text Available Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP. Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

  18. Differential gene expression for Curvularia eragrostidis pathogenic incidence in crabgrass (Digitaria sanguinalis) revealed by cDNA-AFLP analysis.

    Science.gov (United States)

    Wang, Jianshu; Wang, Xuemin; Yuan, Bohua; Qiang, Sheng

    2013-01-01

    Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

  19. Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA.

    Science.gov (United States)

    Yadav, Arun A; Wu, Xing; Patel, Daywin; Yalowich, Jack C; Hasinoff, Brian B

    2014-11-01

    Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI(50) in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

  20. Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (η) in pathogenic yeast Candida albicans

    Science.gov (United States)

    Satpati, Suresh; Manohar, Kodavati; Acharya, Narottam; Dixit, Anshuman

    2017-01-01

    Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

  1. Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes.

    Science.gov (United States)

    Ye, Y W; Ling, N; Han, Y J; Wu, Q P

    2014-11-01

    Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10(2)cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28 °C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.

  2. A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers.

    Science.gov (United States)

    Moayed, Fatemeh; Mashaghi, Alireza; Tans, Sander J

    2013-01-01

    Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

  3. A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers.

    Directory of Open Access Journals (Sweden)

    Fatemeh Moayed

    Full Text Available Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN and the peptide StrepTag II (ST. We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST. In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV linkage. It can be used in conjunction with Neutravidin (NTV-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

  4. Label-Free Dengue Detection Utilizing PNA/DNA Hybridization Based on the Aggregation Process of Unmodified Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Samsulida Abdul Rahman

    2014-01-01

    Full Text Available A label-free optical detection method based on PNA/DNA hybridization using unmodified gold nanoparticles (AuNPs for dengue virus detection has been successfully developed. In this study, no immobilization method is involved and the hybridization of PNA/DNA occurs directly in solution. Unmodified AuNPs undergo immediate aggregation in the presence of neutral charge peptide nucleic acid (PNA due to the coating of PNA on AuNPs surface. However, in the presence of complementary targets DNA, the hybridization of PNA probe with target DNA forms negatively charged complexes due to the negatively charged phosphate backbone of the target DNA. The negatively charged complexes adsorbed onto the AuNPs surface ensure sufficient charge repulsion, need for AuNPs dispersion, and stability in solution. The detection procedure is a naked eye method based on immediate color changes and also through UV-vis adsorption spectra. The selectivity of the proposed method was studied successfully by single base mismatch and noncomplementary target DNA.

  5. A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

    Science.gov (United States)

    Tans, Sander J.

    2013-01-01

    Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications. PMID:23336001

  6. Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species.

    Science.gov (United States)

    Giles, Timothy; Yon, Lisa; Hannant, Duncan; Barrow, Paul; Abu-Median, Abu-Bakr

    2015-12-01

    The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species.

  7. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  8. QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV.

    Science.gov (United States)

    Hong, Sung-Rok; Jeong, Hyun-Do; Hong, Suhee

    2010-08-15

    Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3' end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin-biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016muM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.

  9. Controlled reduction of photobleaching in DNA origami-gold nanoparticle hybrids.

    Science.gov (United States)

    Pellegrotti, Jesica V; Acuna, Guillermo P; Puchkova, Anastasiya; Holzmeister, Phil; Gietl, Andreas; Lalkens, Birka; Stefani, Fernando D; Tinnefeld, Philip

    2014-05-14

    The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles' size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching.

  10. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Heinz-Ulrich G. Weier

    2012-12-01

    Full Text Available Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

  11. DNA metalating-intercalating hybrid agents for the treatment of chemoresistant cancers.

    Science.gov (United States)

    Suryadi, Jimmy; Bierbach, Ulrich

    2012-10-08

    Nonclassical platinum-based antitumor agents have shown enormous potential in the treatment of chemoresistant cancers. The design of these agents is based on the hypothesis that platinum-containing pharmacophores that react with nuclear DNA in cancer cells radically differently than the clinical agent cisplatin will produce a unique spectrum of biological activity. One such class of molecules are platinum-acridine hybrid agents derived from the prototypical complex [PtCl(en)(ACRAMTU)](NO(3))(2), en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea ("PT-ACRAMTU"). This article summarizes milestones in the development of these agents and reviews critical key concepts that have guided their design and that of related compounds. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    Science.gov (United States)

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides.

  13. Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.

    Science.gov (United States)

    Totsingan, Filbert; Bell, Anthony J

    2013-11-01

    The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg(2+), low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg(2+). 4WJ-PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2), while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (K(D)s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

  14. Assembly fabrication of linkers on glass surface and their effect on DNA synthesis and hybridization

    Institute of Scientific and Technical Information of China (English)

    ShenJiayao; XiaoPengfeng; HouPeng; JiMeiju; SunXiao; HeNongyue

    2003-01-01

    Linkers were assembled on a glass surface based on the hydrolysis and condensation of 3-glycidoxy-propyltrimethoxysilane (GPS). After the assembly of GPS, four approaches were tried to open the ending epoxide group of GPS or to further elongate the linkers. The effect of these approaches on DNA in situ synthesis and hybridization was investigated. For the spacing of the synthesis initiation sites, the wettability of the support and the length of the linking group that attaches the initiation site to the surface have direct influences on the yield of coupling reactions and the subsequent hybridization events. X-ray photoelectron spectroscopy (XPS) and mean contact angles of deionized water of the above slides were measured to assess the linker's characteristics in each procedure. It was proved that the glass slides were successfully modified and became excellent supports for the oligonucleotides synthesis. In addition, it proved best for the in situ oligonueleotides synthesis that a glass slide was in turn treated with ethylenediamine, glutaradehyde, ethanolamine and sodium borohydride solution at ambient temperature after silanized with GPS.

  15. Morphology vs Genetics: the hybrid origin of a sea turtle disproved by DNA

    Directory of Open Access Journals (Sweden)

    L. GAROFALO

    2013-01-01

    Full Text Available A putative hybrid sea turtle juvenile was evaluated with discriminant DNA markers. When compared with standard values for sea turtles, the general morphological features assigned the specimen to Caretta caretta, while the shape and coloration of the head and the beak profile fell within the Eretmochelys imbricata range; the front flippers were instead like those of a Chelonia mydas. Moreover, prefrontal scale number was outside the putative parental species’ ranges. The mitochondrial D-loop sequence was from C. caretta, and matched haplotype CC-A2.1, the most common in the Mediterranean. Sequence profiles at three nuclear loci withspecies-specific substitutions (Cmos, BDNF and R35 revealed only C. caretta variants, thus excluding that the individual wasan F1 hybrid. This study highlights the importance of integrating different methodological approaches to understand reproductive animal biology and to set the boundaries for specific morphological traits. In particular, we propose the genetic analysis of a new combination of mitochondrial and nuclear markers as a standard procedure which can be adopted in the identification of sea turtlehybrids.

  16. Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA--thermodynamic studies.

    Science.gov (United States)

    Nieborak, Anna; Górecki, Andrzej

    2016-03-01

    This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF.

  17. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used...... typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars...

  18. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids

    Science.gov (United States)

    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E.; Pandya, Unnati M.

    2017-01-01

    Abstract Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. PMID:28334836

  19. Molecular cytogenetics of Alstroemeria: identification of parental genomes in interspecific hybrids and characterization of repetitive DNA families in constitutive heterochromatin.

    Science.gov (United States)

    Kuipers, A G; van Os, D P; de Jong, J H; Ramanna, M S

    1997-02-01

    The genus Alstroemeria consists of diploid (2n = 2x = 16) species originating mainly from Chile and Brazil. Most cultivars are triploid or tetraploid interspecific hybrids. C-banding of eight species revealed obvious differentiation of constitutive heterochromatin within the genus. The present study focused on the molecular (cyto)genetic background of this differentiation. Genomic slot-blot analysis demonstrated strong conservation of major parts of the genomes among six species. The chromosomes of A. aurea and A. ligtu, species with pronounced interstitial C-bands, were found to contain large amounts of highly repetitive and species-specific DNA. The variation in size, number and intensity of strongly probed bands of major repetitive DNA families observed in genomic Southern blots of Sau3A, HaeIII, and MseI digests indicated a strong correlation between variation in genomic DNA composition and different C-banding patterns among Alstroemeria species. Genomic in situ hybridization (GISH) revealed a clear distinction between parental chromosomes in the hybrids between Chilean and Brazilian species and also between Chilean species, as long as at least one of the parental species possessed prominent C-banding. Regarding the latter, discriminative hybridization resulted from highly repetitive species specific DNA in the heterochromatic chromosome regions of A. aurea and A. ligtu, and caused GISH banding patterns that coincided with the C-banding patterns.

  20. Not just "a clever way to detect whether DNA really made RNA": The invention of DNA-RNA hybridization and its outcome.

    Science.gov (United States)

    Fisher, Susie

    2015-10-01

    The invention of DNA-RNA hybridization in 1960 by Ben Hall and Sol Spiegelman had a powerful impact on the theory and discourse of molecular biology. Yet, despite its importance, the story of this invention has barely been told. Hybridization allowed biologists to bridge the theoretical realm and the material world of organisms, to correlate a hypothetical concept of biological information transfer with a mechanism capable of making an RNA copy of DNA. During the early 1960s, Spiegelman and coworkers employed hybridization to investigate the origin of RNAs found in cells. They operationally defined messenger RNA and elucidated several aspects of genome organization. For Spiegelman, this was the culmination of his longstanding interest in the mechanism of enzyme/protein synthesis; for Hall, it was the beginning of a successful career in genetics. Other scientists immediately recognized the power of the technique and introduced improvements. In 1965, Gillespie and Spiegelman combined several modifications and described a procedure for hybridization that became standard. Since the 1970s, it has become an essential tool in biology and in biotechnology, and a core component in molecular techniques such as DNA microarrays. Notwithstanding its current success, the inventors' names have disappeared from the literature. This curiosity is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  2. Analysis of nuclear and organellar DNA in somatic hybrids between solanaceous species.

    NARCIS (Netherlands)

    Wolters, A.M.A.

    1994-01-01

    This thesis describes an analysis of the possibilities and limitations of somatic hybridization of solanaceous species. Emphasis was laid on the elucidation of the interactions between nuclei, chloroplasts and mitochondria in the obtained somatic hybrids. Hybridization experiments between tomato ( L

  3. HyDEn: a hybrid steganocryptographic approach for data encryption using randomized error-correcting DNA codes.

    Science.gov (United States)

    Tulpan, Dan; Regoui, Chaouki; Durand, Guillaume; Belliveau, Luc; Léger, Serge

    2013-01-01

    This paper presents a novel hybrid DNA encryption (HyDEn) approach that uses randomized assignments of unique error-correcting DNA Hamming code words for single characters in the extended ASCII set. HyDEn relies on custom-built quaternary codes and a private key used in the randomized assignment of code words and the cyclic permutations applied on the encoded message. Along with its ability to detect and correct errors, HyDEn equals or outperforms existing cryptographic methods and represents a promising in silico DNA steganographic approach.

  4. Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing.

    Science.gov (United States)

    Hu, Yuwei; Hua, Shucheng; Li, Fenghua; Jiang, Yuanyuan; Bai, Xiaoxue; Li, Dan; Niu, Li

    2011-07-15

    Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged -COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH₂-IL) was applied to the reduction of HAuCl₄ to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH₂-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.

  5. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    Science.gov (United States)

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric

    2016-02-24

    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.

  6. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.

    Science.gov (United States)

    Fiegler, Heike; Carr, Philippa; Douglas, Eleanor J; Burford, Deborah C; Hunt, Sarah; Scott, Carol E; Smith, James; Vetrie, David; Gorman, Patricia; Tomlinson, Ian P M; Carter, Nigel P

    2003-04-01

    We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet.

  7. Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

    Science.gov (United States)

    Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2017-06-01

    Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

  8. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  9. Hybrid Taguchi DNA Swarm Intelligence for Optimal Inverse Kinematics Redundancy Resolution of Six-DOF Humanoid Robot Arms

    Directory of Open Access Journals (Sweden)

    Hsu-Chih Huang

    2014-01-01

    Full Text Available This paper presents a hybrid Taguchi deoxyribonucleic acid (DNA swarm intelligence for solving the inverse kinematics redundancy problem of six degree-of-freedom (DOF humanoid robot arms. The inverse kinematics problem of the multi-DOF humanoid robot arm is redundant and has no general closed-form solutions or analytical solutions. The optimal joint configurations are obtained by minimizing the predefined performance index in DNA algorithm for real-world humanoid robotics application. The Taguchi method is employed to determine the DNA parameters to search for the joint solutions of the six-DOF robot arms more efficiently. This approach circumvents the disadvantage of time-consuming tuning procedure in conventional DNA computing. Simulation results are conducted to illustrate the effectiveness and merit of the proposed methods. This Taguchi-based DNA (TDNA solver outperforms the conventional solvers, such as geometric solver, Jacobian-based solver, genetic algorithm (GA solver and ant, colony optimization (ACO solver.

  10. Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis

    DEFF Research Database (Denmark)

    Jensen, Annette B; Welker, Dennis L; Kryger, Per

    2012-01-01

    was then compared among the different loci, and three were found to have the greatest detection power for identifying A. apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host–pathogen interaction and to test evolutionary hypotheses...

  11. Interaction between root-knot nematodes and Solanum spp. Variation in pathogenicity, cytology, proteins and DNA.

    NARCIS (Netherlands)

    Beek, van der J.G.

    1997-01-01

    This thesis describes genetic variation in the root-knot nematodes Meloidogyne hapla, M. chitwoodi and M. fallax, particularly with respect to their pathogenicity on Solanum spp. Significant differences in virulence and aggressiveness were shown to exist between and within these species. Evidence fo

  12. Chemical Composition, Antioxidant, DNA Damage Protective, Cytotoxic and Antibacterial Activities of Cyperus rotundus Rhizomes Essential Oil against Foodborne Pathogens

    Science.gov (United States)

    Hu, Qing-Ping; Cao, Xin-Ming; Hao, Dong-Lin; Zhang, Liang-Liang

    2017-01-01

    Cyperus rotundus L. (Cyperaceae) is a medicinal herb traditionally used to treat various clinical conditions at home. In this study, chemical composition of Cyperus rotundus rhizomes essential oil, and in vitro antioxidant, DNA damage protective and cytotoxic activities as well as antibacterial activity against foodborne pathogens were investigated. Results showed that α-cyperone (38.46%), cyperene (12.84%) and α-selinene (11.66%) were the major components of the essential oil. The essential oil had an excellent antioxidant activity, the protective effect against DNA damage, and cytotoxic effects on the human neuroblastoma SH-SY5Y cell, as well as antibacterial activity against several foodborne pathogens. These biological activities were dose-dependent, increasing with higher dosage in a certain concentration range. The antibacterial effects of essential oil were greater against Gram-positive bacteria as compared to Gram-negative bacteria, and the antibacterial effects were significantly influenced by incubation time and concentration. These results may provide biological evidence for the practical application of the C. rotundus rhizomes essential oil in food and pharmaceutical industries. PMID:28338066

  13. DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

    Science.gov (United States)

    Bronder, Thomas S; Poghossian, Arshak; Scheja, Sabrina; Wu, Chunsheng; Keusgen, Michael; Mewes, Dieter; Schöning, Michael J

    2015-09-16

    Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

  14. A rapid method for isolation of total DNA from pathogenic filamentous plant fungi.

    Science.gov (United States)

    González-Mendoza, D; Argumedo-Delira, R; Morales-Trejo, A; Pulido-Herrera, A; Cervantes-Díaz, L; Grimaldo-Juarez, O; Alarcón, A

    2010-02-02

    DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A(260/280) absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A(260/230) values were higher than 1.8, suggesting negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction.

  15. cDNA-AFLP analysis of plant and pathogen genes expressed in grapevine infected with Plasmopara viticola

    Directory of Open Access Journals (Sweden)

    Kortekamp Andreas

    2008-03-01

    Full Text Available Abstract Background The oomycete Plasmopara viticola (Berk. and Curt. Berl. and de Toni causes downy mildew in grapevine (Vitis vinifera L.. This pathogen is strictly biotrophic, thus completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a large-scale cDNA-AFLP analysis to identify grapevine and P. viticola genes associated with the infection process. Results We carried out cDNA-AFLP analysis on artificially infected leaves of the susceptible cultivar Riesling at the oil spot stage, on water-treated leaves and on a sample of pure sporangia as controls. Selective amplifications with 128 primer combinations allowed the visualization of about 7000 transcript-derived fragments (TDFs in infected leaves, 1196 of which (17% were differentially expressed. We sequenced 984 fragments, 804 of which were identified as grapevine transcripts after homology searching, while 96 were homologous to sequences in Phytophthora spp. databases and were attributed to P. viticola. There were 82 orphan TDFs. Many grapevine genes spanning almost all functional categories were downregulated during infection, especially genes involved in photosynthesis. Grapevine genes homologous to known resistance genes also tended to be repressed, as were several resistance gene analogs and carbonic anhydrase (recently implicated in pathogen resistance. In contrast, genes encoding cytoskeletal components, enzymes of the phenylpropanoid and beta-oxidation pathways, and pathogenesis related proteins were primarily upregulated during infection. The majority of P. viticola transcripts expressed in planta showed homology to genes of unknown function or to genomic Phytophthora sequences, but genes related to metabolism, energy production, transport and signal transduction were also identified. Conclusion This study provides the first global catalogue of grapevine and P

  16. Genomic and Transcriptomic Analyses of Foodborne Bacterial Pathogens

    Science.gov (United States)

    Zhang, Wei; Dudley, Edward G.; Wade, Joseph T.

    DNA microarrays (often interchangeably called DNA chips or DNA arrays) are among the most popular analytical tools for high-throughput comparative genomic and transcriptomic analyses of foodborne bacterial pathogens. A typical DNA microarray contains hundreds to millions of small DNA probes that are chemically attached (or "printed") onto the surface of a microscopic glass slide. Depending on the specific "printing" and probe synthesis technologies for different microarray platforms, such DNA probes can be PCR amplicons or in situ synthesized short oligonucleotides. DNA microarray technologies have revolutionized the way that we investigate the biology of foodborne bacterial pathogens. The major advantage of these technologies is that DNA microarrays allow comparison of subtle genomic or transcriptomic variations between two bacterial samples, such as genomic variations between two different bacterial strains or transcriptomic alterations of same bacterial strain under two different treatments. Some applications of comparative genomic hybridization microarrays and global gene expression microarrays have been covered in previous chapters of this book.

  17. Recent introgressive hybridization revealed by exclusive mtDNA transfer from Oreochromis leucostictus (Trewavas, 1933) to Oreochromis niloticus (Linnaeus, 1758) in Lake Baringo, Kenya

    OpenAIRE

    Nyingi, Dorothy W.; Agnèse, Jean-François

    2007-01-01

    Nuclear DNA and mtDNA polymorphisms were surveyed in various species of East African Oreochromis. In Lake Baringo, where only Oreochromis niloticus baringoensis is present, alien mtDNA haplotypes were observed, apparently the result of introgressive hybridization with Oreochromis leucostictus. This introgression is not accompanied by any substantial or recorded transfer of nuclear genes into O. n. baringoensis.

  18. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    Science.gov (United States)

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-12-10

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

  19. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences.

    Science.gov (United States)

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen

    2012-03-23

    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  20. Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice.

    Directory of Open Access Journals (Sweden)

    Srinivas Rao

    Full Text Available BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.

  1. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    Science.gov (United States)

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  2. 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.

  3. A controlled microfluidic electrochemical lab-on-a-chip for label-free diffusion-restricted DNA hybridization analysis.

    Science.gov (United States)

    Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

    2015-02-15

    Lab-on-a-chip (LOC) devices for electrochemical analysis of DNA hybridization events offer a technology for real-time and label-free assessment of biomarkers at the point-of-care. Here, we present a microfluidic LOC, with 3 × 3 arrayed electrochemical sensors for the analysis of DNA hybridization events. A new dual layer microfluidic valved manipulation system is integrated providing controlled and automated capabilities for high throughput analysis. This feature improves the repeatability, accuracy, and overall sensing performance (Fig. 1). The electrochemical activity of the fabricated microfluidic device is validated and demonstrated repeatable and reversible Nernstian characteristics. System design required detailed analysis of energy storage and dissipation as our sensing modeling involves diffusion-related electrochemical impedance spectroscopy. The effect of DNA hybridization on the calculated charge transfer resistance and the diffusional resistance components is evaluated. We demonstrate a specific device with an average cross-reactivity value of 27.5%. The device yields semilogarithmic dose response and enables a theoretical detection limit of 1 nM of complementary ssDNA target. This limit is lower than our previously reported non-valved device by 74% due to on-chip valve integration providing controlled and accurate assay capabilities.

  4. Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

    Science.gov (United States)

    Xu, Shicai; Zhan, Jian; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Gao, Shoubao; Guo, Chengang; Liu, Hanping; Li, Zhenhua; Wang, Jihua; Zhou, Yaoqi

    2017-03-01

    Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.

  5. Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

    Science.gov (United States)

    Xu, Shicai; Zhan, Jian; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Gao, Shoubao; Guo, Chengang; Liu, Hanping; Li, Zhenhua; Wang, Jihua; Zhou, Yaoqi

    2017-01-01

    Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array. PMID:28322227

  6. Special DNA Methylated Sites Between Haploid of Twin-Seedling and Its Hybrids in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    WU Shao-hua; XUE Jing-jing; ZHANG Hong-yu; XU Pei-zhou; WU Xian-jun

    2012-01-01

    DNA methylation is one of the epigenetic phenomena which can be transferred to the offspring by cell division in the evolution of organisms.The epigenetic regulation accompanied by gene expression can be found directly in the phenotype of haploidy plants.DNA cytosine methylation at the 5'-CpCpGpG sites of haploid,Shuhui 527,Shuhui 363 and their hybrids was analyzed by methylation sensitive amplification polymorphism (MSAP) method.There were 765 DNA methylated sites detected and the methylation level was lower in hybrids than parents.Meanwhile,the different bands between hybrids and parents were analyzed and two types of methylated sites were detected,of which one inherited from haploid,and the other did not.The biological functions of genes related to methylated sites involved in cell structure,metabolize and response factor.Therefore,DNA methylated modifications can activate and silence the genes and play an important role in plant growth,development and evolution.

  7. 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.

  8. DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple.

    Science.gov (United States)

    O'Gorman, Daniel T; Sholberg, Peter L; Stokes, Sarah C; Ginns, J

    2008-01-01

    The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.

  9. Comparison of rapid hybridization-based pathogen identification and resistance evaluation in sepsis using the Verigene® device paired with "good old culture".

    Science.gov (United States)

    Berktold, Michael; Mutschlechner, Wolfgang; Orth-Höller, Dorothea

    2017-06-01

    Rapid microbial diagnostics is important for septicemic patients. The current gold standard is blood culture with consecutive pathogen identification and antimicrobial susceptibility testing. However, these culture-based methods need at least 48 h.The aim of this study was to compare Verigene(®) (Nanosphere, Northbrook, IL, USA), a rapid hybridization-based method, with conventional culture-based methods for detection of pathogens and resistance markers from positive blood cultures of septic patients.In 85 of 100 tested blood culture samples (85 %), pathogen identification as well as resistance profile were identical in Verigene and conventional culture. In 4 %, discordant results were observed. In 9 %, conventional culture revealed a pathogen ID or resistance phenotype not included in the Verigene panel. In 2 % no Verigene result was available.In conclusion, Verigene offers the availability of fast and reliable pathogen identification and resistance profile determination, which may result in an earlier start of adequate antimicrobial treatment.

  10. Dorsal horn-enriched genes identified by DNA microarray, in situ hybridization and immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Koblan Kenneth S

    2002-08-01

    Full Text Available Abstract Background Neurons in the dorsal spinal cord play important roles in nociception and pain. These neurons receive input from peripheral sensory neurons and then transmit the signals to the brain, as well as receive and integrate descending control signals from the brain. Many molecules important for pain transmission have been demonstrated to be localized to the dorsal horn of the spinal cord. Further understanding of the molecular interactions and signaling pathways in the dorsal horn neurons will require a better knowledge of the molecular neuroanatomy in the dorsal spinal cord. Results A large scale screening was conducted for genes with enriched expression in the dorsal spinal cord using DNA microarray and quantitative real-time PCR. In addition to genes known to be specifically expressed in the dorsal spinal cord, other neuropeptides, receptors, ion channels, and signaling molecules were also found enriched in the dorsal spinal cord. In situ hybridization and immunohistochemistry revealed the cellular expression of a subset of these genes. The regulation of a subset of the genes was also studied in the spinal nerve ligation (SNL neuropathic pain model. In general, we found that the genes that are enriched in the dorsal spinal cord were not among those found to be up-regulated in the spinal nerve ligation model of neuropathic pain. This study also provides a level of validation of the use of DNA microarrays in conjunction with our novel analysis algorithm (SAFER for the identification of differences in gene expression. Conclusion This study identified molecules that are enriched in the dorsal horn of the spinal cord and provided a molecular neuroanatomy in the spinal cord, which will aid in the understanding of the molecular mechanisms important in nociception and pain.

  11. Chromosome segregation in Archaea mediated by a hybrid DNA partition machine.

    Science.gov (United States)

    Kalliomaa-Sanford, Anne K; Rodriguez-Castañeda, Fernando A; McLeod, Brett N; Latorre-Roselló, Victor; Smith, Jasmine H; Reimann, Julia; Albers, Sonja V; Barillà, Daniela

    2012-03-06

    Eukarya and, more recently, some bacteria have been shown to rely on a cytoskeleton-based apparatus to drive chromosome segregation. In contrast, the factors and mechanisms underpinning this fundamental process are underexplored in archaea, the third domain of life. Here we establish that the archaeon Sulfolobus solfataricus harbors a hybrid segrosome consisting of two interacting proteins, SegA and SegB, that play a key role in genome segregation in this organism. SegA is an ortholog of bacterial, Walker-type ParA proteins, whereas SegB is an archaea-specific factor lacking sequence identity to either eukaryotic or bacterial proteins, but sharing homology with a cluster of uncharacterized factors conserved in both crenarchaea and euryarchaea, the two major archaeal sub-phyla. We show that SegA is an ATPase that polymerizes in vitro and that SegB is a site-specific DNA-binding protein contacting palindromic sequences located upstream of the segAB cassette. SegB interacts with SegA in the presence of nucleotides and dramatically affects its polymerization dynamics. Our data demonstrate that SegB strongly stimulates SegA polymerization, possibly by promoting SegA nucleation and accelerating polymer growth. Increased expression levels of segAB resulted in severe growth and chromosome segregation defects, including formation of anucleate cells, compact nucleoids confined to one half of the cell compartment and fragmented nucleoids. The overall picture emerging from our findings indicates that the SegAB complex fulfills a crucial function in chromosome segregation and is the prototype of a DNA partition machine widespread across archaea.

  12. M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity

    Science.gov (United States)

    Park, Hyung-Youl; Dugasani, Sreekantha Reddy; Kang, Dong-Ho; Yoo, Gwangwe; Kim, Jinok; Gnapareddy, Bramaramba; Jeon, Jaeho; Kim, Minwoo; Song, Young Jae; Lee, Sungjoo; Heo, Jonggon; Jeon, Young Jin; Park, Sung Ha; Park, Jin-Hong

    2016-01-01

    Here, we report a high performance biosensor based on (i) a Cu2+-DNA/MoS2 hybrid structure and (ii) a field effect transistor, which we refer to as a bio-FET, presenting a high sensitivity of 1.7 × 103 A/A. This high sensitivity was achieved by using a DNA nanostructure with copper ions (Cu2+) that induced a positive polarity in the DNA (receptor). This strategy improved the detecting ability for doxorubicin-like molecules (target) that have a negative polarity. Very short distance between the biomolecules and the sensor surface was obtained without using a dielectric layer, contributing to the high sensitivity. We first investigated the effect of doxorubicin on DNA/MoS2 and Cu2+-DNA/MoS2 nanostructures using Raman spectroscopy and Kelvin force probe microscopy. Then, we analyzed the sensing mechanism and performance in DNA/MoS2- and Cu2+-DNA/MoS2-based bio-FETs by electrical measurements (ID-VG at various VD) for various concentrations of doxorubicin. Finally, successful operation of the Cu2+-DNA/MoS2 bio-FET was demonstrated for six cycles (each cycle consisted of four steps: 2 preparation steps, a sensing step, and an erasing step) with different doxorubicin concentrations. The bio-FET showed excellent reusability, which has not been achieved previously in 2D biosensors. PMID:27775004

  13. The formation of diploid and triploid hybrids of female grass carp × male blunt snout bream and their 5S rDNA analysis.

    Science.gov (United States)

    He, Weiguo; Xie, Lihua; Li, Tangluo; Liu, Shaojun; Xiao, Jun; Hu, Jie; Wang, Jing; Qin, Qinbo; Liu, Yun

    2013-11-23

    Hybridization is a useful strategy to alter the genotypes and phenotypes of the offspring. It could transfer the genome of one species to another through combing the different genome of parents in the hybrid offspring. And the offspring may exhibit advantages in growth rate, disease resistance, survival rate and appearance, which resulting from the combination of the beneficial traits from both parents. Diploid and triploid hybrids of female grass carp (Ctenopharyngodon idellus, GC, Cyprininae, 2n = 48) × male blunt snout bream (Megalobrama amblycephala, BSB, Cultrinae, 2n = 48) were successfully obtained by distant hybridization. Diploid hybrids had 48 chromosomes, with one set from GC and one set from BSB. Triploid hybrids possessed 72 chromosomes, with two sets from GC and one set from BSB.The morphological traits, growth rates, and feeding ecology of the parents and hybrid offspring were compared and analyzed. The two kinds of hybrid offspring exhibited significantly phenotypic divergence from GC and BSB. 2nGB hybrids showed similar growth rate compared to that of GC, and 3nGB hybrids significantly higher results. Furthermore, the feeding ecology of hybrid progeny was omnivorous.The 5S rDNA of GC, BSB and their hybrid offspring were also cloned and sequenced. There was only one type of 5S rDNA (designated type I: 180 bp) in GC and one type of 5S rDNA (designated type II: 188 bp) in BSB. However, in the hybrid progeny, diploid and triploid hybrids both inherited type I and type II from their parents, respectively. In addition, a chimera of type I and type II was observed in the genome of diploid and triploid hybrids, excepting a 10 bp of polyA insertion in type II sequence of the chimera of the diploid hybrids. This is the first report of diploid and triploid hybrids being produced by crossing GC and BSB, which have the same chromosome number. The obtainment of two new hybrid offspring has significance in fish genetic breeding. The results illustrate the effect

  14. A hybrid swarm population of Pinus densiflora × P. sylvestris inferred from sequence analysis of chloroplast DNA and morphological characters

    Institute of Scientific and Technical Information of China (English)

    Young Hee Joung; Jerry L.Hill; Jung Oh Hyun; Ding Mu; Juchun Luo; Do Hyung Lee; Takayuki Kawahara; Jeung Keun Suh; Mark S.Roh

    2013-01-01

    To confirm a hybrid swarm population ofPinus densiflora × P.sylvestris in Jilin,China,we used needles and seeds from P.densiflora,P.sylvestris,and P.densiflora × P.sylvestris collected from natural stands or experimental stations to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker (cpDNA SSRs).Total genomic DNA was extracted and subjected to sequence analysis of the pine cpDNA SSR marker Pt15169.Results show that morphological characters from 4-year old seedlings did not correlate with sequence variants of this marker.Marker haplotypes from all P.sylvestris trees had a CTAT element that was absent from all sampled P.densiflora trees.However,both haplotype classes involving this insertion/deletion element were found in a P.densiflora × P.sylvestris population and its seedling progeny.It was concluded that the P.densiflora × P.sylvestris accessions sampled from Jilin,China resulted from bi-directional crosses,as evidenced by both species' cpDNA haplotypes within the hybrid swarm population.

  15. Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization

    Directory of Open Access Journals (Sweden)

    R. Mezzanotte

    2010-01-01

    Full Text Available The genome of stallion (Spanish breed and donkey (Spanish endemic Zamorano-Leonés were compared using whole comparative genomic in situ hybridization (W-CGH technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

  16. Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization

    Directory of Open Access Journals (Sweden)

    J. Gosalvez

    2010-01-01

    Full Text Available The genome of stallion (Spanish breed and donkey (Spanish endemic Zamorano-Leonés were compared using whole comparative genomic in situ hybridization (W-CGH technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

  17. A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    John C. Leach

    2016-03-01

    Full Text Available The side effects of radio- and chemo-therapy pose long-term challenges on a cancer patient’s health. It is, therefore, highly desirable to develop more effective therapies that can specifically target carcinoma cells without damaging normal and healthy cells. Tremendous efforts have been made in the past to develop targeted drug delivery systems for solid cancer treatment. In this study, a new aptamer, A10-3-J1, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA, was designed. A super paramagnetic iron oxide nanoparticle-aptamer-doxorubicin (SPIO-Apt-Dox was fabricated and employed as a targeted drug delivery platform for cancer therapy. This DNA RNA hybridized aptamer antitumor agent was able to enhance the cytotoxicity of targeted cells while minimizing collateral damage to non-targeted cells. This SPIO-Apt-Dox nanoparticle has specificity to PSMA+ prostate cancer cells. Aptamer inhibited nonspecific uptake of membrane-permeable doxorubic to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake while enhancing targeted cytotoxicity and endocytic uptake. The experimental results indicate that the drug delivery platform can yield statistically significant effectiveness being more cytotoxic to the targeted cells as opposed to the non-targeted cells.

  18. Influence of perylenediimide–pyrene supramolecular interactions on the stability of DNA-based hybrids: Importance of electrostatic complementarity

    Directory of Open Access Journals (Sweden)

    Christian B. Winiger

    2014-07-01

    Full Text Available Aromatic π–π stacking interactions are ubiquitous in nature, medicinal chemistry and materials sciences. They play a crucial role in the stacking of nucleobases, thus stabilising the DNA double helix. The following paper describes a series of chimeric DNA–polycyclic aromatic hydrocarbon (PAH hybrids. The PAH building blocks are electron-rich pyrene and electron-poor perylenediimide (PDI, and were incorporated into complementary DNA strands. The hybrids contain different numbers of pyrene–PDI interactions that were found to directly influence duplex stability. As the pyrene–PDI ratio approaches 1:1, the stability of the duplexes increases with an average value of 7.5 °C per pyrene–PDI supramolecular interaction indicating the importance of electrostatic complementarity for aromatic π–π stacking interactions.

  19. A solid-state electrochemiluminescence biosensing switch for detection of DNA hybridization based on ferrocene-labeled molecular beacon

    Energy Technology Data Exchange (ETDEWEB)

    Wang Xiaoying [School of Public Health, Southeast University, Nanjing 210009 (China); He Pingang, E-mail: pghe@chem.ecnu.edu.c [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Fang Yuzhi, E-mail: yzfang@chem.ecnu.edu.c [Department of Chemistry, East China Normal University, Shanghai 200062 (China)

    2010-08-15

    A solid-state electrochemiluminescence (ECL) biosensing switch incorporating quenching of ECL of ruthenium(II) tris-(bipyridine) (Ru(bpy){sub 3}{sup 2+}) by ferrocene (Fc) has been successfully developed for DNA hybridization detection. The important issue for this biosensing system is based on the ferrocene-labeled molecular beacon (Fc-MB), i.e. using the special Fc-MB to react with the target DNA and then change its structure, resulting in an ECL intensity change. Under the optimal conditions, the difference of ECL intensity before and after the hybridization reaction ({Delta}I{sub ECL}) was linearly related to the concentration of the complementary sequence in the range of 10 fM-10 pM and the detection limit was down to 1.0 fM.

  20. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    Science.gov (United States)

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  1. Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer%麝致病性大肠杆菌的质粒DNA分析

    Institute of Scientific and Technical Information of China (English)

    罗燕; 程建国; 郑士华; 赵翠; 李蓓; 李敏

    2009-01-01

    [Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.

  2. Identification of DNA sequences from a second pathogenicity island of uropathogenic Escherichia coli CFT073: probes specific for uropathogenic populations.

    Science.gov (United States)

    Rasko, D A; Phillips, J A; Li, X; Mobley, H L

    2001-10-15

    Uropathogenic Escherichia coli is the leading cause of urinary tract infection and hospital visits in North America. Cystitis and acute pyelonephritis, infection of the bladder and kidney, respectively, are the two most common syndromes encountered in patients with urinary tract infection. We sequenced and annotated 71,684 bases of a previously unidentified pathogenicity-associated island (PAI) from E. coli strain CFT073. This PAI contained 89 open-reading frames encoding a pap operon, iron-regulated genes, mobile genetic elements, and a large proportion of unknown or unidentified open-reading frames. Dot blot analysis with 11 DNA sequences from this PAI demonstrated that 7 sequences were more prevalent among uropathogens: 2 probes were more prevalent among cystitis and pyelonephritis isolates, 2 among pyelonephritis isolates only, and 3 among cystitis isolates only than among fecal isolates. These data suggest that groups of uropathogens have genetic differences that may be responsible for the different clinical outcomes.

  3. 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...

  4. Cutting edge: natural DNA repetitive extragenic sequences from gram-negative pathogens strongly stimulate TLR9.

    Science.gov (United States)

    Magnusson, Mattias; Tobes, Raquel; Sancho, Jaime; Pareja, Eduardo

    2007-07-01

    Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-alpha production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9(tm1Aki) knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.

  5. Finding a human telomere DNA-RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: a protective structure for telomere end.

    Science.gov (United States)

    Xu, Yan; Suzuki, Yuta; Ishizuka, Takumi; Xiao, Chao-Da; Liu, Xiao; Hayashi, Tetsuya; Komiyama, Makoto

    2014-08-15

    Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

  6. UMD‐Predictor: A High‐Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution

    Science.gov (United States)

    Salgado, David; Desvignes, Jean‐Pierre; Rai, Ghadi; Blanchard, Arnaud; Miltgen, Morgane; Pinard, Amélie; Lévy, Nicolas; Collod‐Béroud, Gwenaëlle

    2016-01-01

    ABSTRACT Whole‐exome sequencing (WES) is increasingly applied to research and clinical diagnosis of human diseases. It typically results in large amounts of genetic variations. Depending on the mode of inheritance, only one or two correspond to pathogenic mutations responsible for the disease and present in affected individuals. Therefore, it is crucial to filter out nonpathogenic variants and limit downstream analysis to a handful of candidate mutations. We have developed a new computational combinatorial system UMD‐Predictor (http://umd‐predictor.eu) to efficiently annotate cDNA substitutions of all human transcripts for their potential pathogenicity. It combines biochemical properties, impact on splicing signals, localization in protein domains, variation frequency in the global population, and conservation through the BLOSUM62 global substitution matrix and a protein‐specific conservation among 100 species. We compared its accuracy with the seven most used and reliable prediction tools, using the largest reference variation datasets including more than 140,000 annotated variations. This system consistently demonstrated a better accuracy, specificity, Matthews correlation coefficient, diagnostic odds ratio, speed, and provided the shortest list of candidate mutations for WES. Webservices allow its implementation in any bioinformatics pipeline for next‐generation sequencing analysis. It could benefit to a wide range of users and applications varying from gene discovery to clinical diagnosis. PMID:26842889

  7. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  8. Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components

    Directory of Open Access Journals (Sweden)

    Marian Filipiak

    2008-03-01

    Full Text Available An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5’-phosphate end to amino group of cysteamine self-assembled monolayer (SAM on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3’- dimethylaminopropyl-carbodiimide (EDC and N-hydroxy-sulfosuccinimide (NHS. The hybridization reaction on the electrode surface was detected via methylene blue (MB presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs give a broad perspectives for analytical application of the biosensor.

  9. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  10. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization

    OpenAIRE

    Germán Robledo; Guillermo Seijo

    2008-01-01

    Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH) of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI) positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with a...

  11. Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

    Science.gov (United States)

    Vuong, Jeni; Collard, Jean-Marc; Whaley, Melissa J.; Bassira, Issaka; Seidou, Issaka; Diarra, Seydou; Ouédraogo, Rasmata T.; Kambiré, Dinanibè; Taylor, Thomas H.; Sacchi, Claudio; Mayer, Leonard W.; Wang, Xin

    2016-01-01

    Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume. PMID:26829233

  12. Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.

    Directory of Open Access Journals (Sweden)

    Jeni Vuong

    Full Text Available Neisseria meningitidis (Nm, Haemophilus influenzae (Hi, and Streptococcus pneumoniae (Sp are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

  13. Behavior of meiotic chromosomes in Pinus wallichiana, P. strobus and their hybrid and nrDNA localization in pollen mother cells of the hybrid by using FISH.

    Science.gov (United States)

    Deng, Hui-Sheng; Zhang, Da-Ming; Fu, Cheng-Xin; Hong, De-Yuan

    2008-03-01

    The complete process of meiosis was investigated in Pinus wallichiana, P. strobus and their artificial hybrid (F1) using microsporocytes. It is revealed that there were slightly lower chiasma frequency, lower ring bivalent frequency, lower meiotic index and distinctly higher frequency of aberrance (chromosomal bridges, fragments or micronuclei) in pollen mother cells (PMCs) of the hybrid (F1) than those of the parental species, which showed a certain degree of differentiation between homologous chromosomes of the two parents. However, relatively higher frequency of ring bivalents and higher meiotic index in all the three entities indicate the great stability of genomes of parental species, and the differentiation of genomes between the two parents must have been slight. Total nineteen signal loci of 18S rDNA were observed in nine bivalents of the hybrid (F1), among which one bivalent bears two loci, while the others have only one. It is suggested that distinct differentiation at genetic level existed in homologous chromosomes of the two parental species, whereas only slight differentiation at karyotypic and genomic levels take place between the parent species.

  14. Behavior of Meiotic Chromosomes in Pinus wallichiana, P. strobus and Their Hybrid and nrDNA Localization in Pollen Mother Cells of the Hybrid by Using FISH

    Institute of Scientific and Technical Information of China (English)

    Hui-Sheng Deng; Da-Ming Zhang; Cheng-Xin Fu; De-Yuan Hong

    2008-01-01

    The complete process of meiosis was investigated in Pinus wallichiana, P. strobus and their artificial hybrid (F1) using microsporocytes. It is revealed that there were slightly lower chiasma frequency, lower ring bivalent frequency, lower meiotic Index and distinctly higher frequency of aberrance (chromosomal bridges, fragments or micronuclei) in pollen mother cells (PMCs) of the hybrid (F1) than those of the parental species, which showed a certain degree of differentiation between homologous chromosomes of the two parents. However, relatively higher frequency of ring bivalents and higher meiotic index in all the three entities indicate the great stability of genomes of parental species, and the differentiation of genomes between the two parents must have been slight. Total nineteen signal loci of 18S rDNA were observed in nine bivalents of the hybrid (F1), among which one bivalent bears two loci, while the others have only one. It is suggested that distinct differentiation at genetic level existed in homologous chromosomes of the two parental species, whereas only slight differentiation at karyotypic and genomic levels take place between the parent species.

  15. 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.

  16. Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANInt) and poly-L-lysine (pLys).The composite of pLys and PANInt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane.The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH,but also possesses fine biocompatibility for bio-analytes.DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity.The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition.The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method.This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)6]3-/4as an indicator.

  17. Probing the Salt Concentration Dependent Nucelobase Distribution in a Single-Stranded DNA-Single-Walled Carbon Nanotube Hybrid with Molecular Dynamics.

    Science.gov (United States)

    Ghosh, Soumadwip; Patel, Nisheet; Chakrabarti, Rajarshi

    2016-01-28

    The hybrids of single-walled carbon nanotube (SWCNT) and single stranded DNA (ssDNA) are novel nanoscale materials having remarkable applications in nanotechnology. The absorption of nucleobases on the surface of a SWCNT depends strongly on the ionic strength of the medium. In this paper, using atomistic molecular dynamics we have shown that at low salt concentration ssDNA wraps on the surface of SWCNT through hydrophobic π-π stacking between the DNA bases and the sp(2)-hybridized carbon atoms of the carbon nanotube. At high salt concentration, however, the DNA molecule adopts a partially folded structure and the ssDNA-SWCNT wrapping gets weakened significantly due to the self-stacking of the DNA bases. Our study can find relevance in CNT mediated gene delivery processes where subsequent unwrapping of the gene from its carrier is anticipated across the cell membrane regulated by an existing salt concentration gradient.

  18. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    Science.gov (United States)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  19. Use of DNA sequencing to detect pathogenic, saprotrophic, and stain fungi in sapwood of declining red pine (Pinus resinosa) in the Upper Midwest

    Science.gov (United States)

    M.T. Banik; D.L. Lindner; J. Juzwik; J.A. Glaeser

    2013-01-01

    An inexpensive kit was developed to collect wood samples for molecular detection of pathogenic, saprotrophic and stain fungi in declining Pinus resinosa in the Upper Midwest. The kit contained materials for "clean" collection of sapwood drill shavings, which were then subjected to PCR of the rDNA ITS region with fungal-specific primers,...

  20. Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers

    Directory of Open Access Journals (Sweden)

    Schneider Horacio

    2011-09-01

    Full Text Available Abstract Background The classical perspective that interspecific hybridization in animals is rare has been changing due to a growing list of empirical examples showing the occurrence of gene flow between closely related species. Using sequence data from cyt b mitochondrial gene and three intron nuclear genes (RPL9, c-myc, and RPL3 we investigated patterns of nucleotide polymorphism and divergence between two closely related toad species R. marina and R. schneideri. By comparing levels of differentiation at nuclear and mtDNA levels we were able to describe patterns of introgression and infer the history of hybridization between these species. Results All nuclear loci are essentially concordant in revealing two well differentiated groups of haplotypes, corresponding to the morphologically-defined species R. marina and R. schneideri. Mitochondrial DNA analysis also revealed two well-differentiated groups of haplotypes but, in stark contrast with the nuclear genealogies, all R. schneideri sequences are clustered with sequences of R. marina from the right Amazon bank (RAB, while R. marina sequences from the left Amazon bank (LAB are monophyletic. An Isolation-with-Migration (IM analysis using nuclear data showed that R. marina and R. schneideri diverged at ≈ 1.69 Myr (early Pleistocene, while R. marina populations from LAB and RAB diverged at ≈ 0.33 Myr (middle Pleistocene. This time of divergence is not consistent with the split between LAB and RAB populations obtained with mtDNA data (≈ 1.59 Myr, which is notably similar to the estimate obtained with nuclear genes between R. marina and R. schneideri. Coalescent simulations of mtDNA phylogeny under the speciation history inferred from nuclear genes rejected the hypothesis of incomplete lineage sorting to explain the conflicting signal between mtDNA and nuclear-based phylogenies. Conclusions The cytonuclear discordance seems to reflect the occurrence of interspecific hybridization between these

  1. Chloroplast DNA variation, postglacial recolonization and hybridization in hazel, Corylus avellana.

    Science.gov (United States)

    Palmé, A E

    2002-09-01

    To unravel the postglacial migration history of hazel, Corylus avellana, the genetic variation at two types of chloroplast DNA markers, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellites, was assessed in 26 natural hazel populations distributed across the range of C. avellana. In addition a sequence of 2468 base pairs, which contains the matK gene, was analysed in seven individuals. Very little variation was detected overall [hT:PCR-RFLP= 0.091, hT:microsatellite= 0.423, pi (nucleotide diversity) = 0.00093] but the microsatellite markers, which have the highest levels of variation, show a clear geographical structure that divides Europe into two areas: (i) Italy and the Balkans, on one hand and (ii) the rest of Europe, on the other hand. These data exclude Italy and the Balkans as possible origins of the postglacial recolonization but cannot unambiguously show which other area is the origin, since the genetic data does not indicate the direction of spread. If we take the pollen record into account, the most likely scenario would be an expansion from southwestern France into most of Europe except Italy and the Balkans, and then a local expansion in the latter area. The two main haplotypes identified with both PCR-RFLP and sequencing, A and B, were found not only in C. avellana but also in other European Corylus species and cultivars. Haplotype A, which is dominating all investigated natural populations of C. avellana, is also found in the European tree hazel (C. colurna) and haplotype B, which is rare in C. avellana, has been identified in the filbert (C. maxima) and C. avellana cultivars. This pattern seems to indicate a history of past hybridization among the European Corylus species and cultivars.

  2. DNA hybridization evidence for the principal lineages of hummingbirds (Aves:Trochilidae).

    Science.gov (United States)

    Bleiweiss, R; Kirsch, J A; Matheus, J C

    1997-03-01

    The spectacular evolutionary radiation of hummingbirds (Trochilidae) has served as a model system for many biological studies. To begin to provide a historical context for these investigations, we generated a complete matrix of DNA hybridization distances among 26 hummingbirds and an outgroup swift (Chaetura pelagica) to determine the principal hummingbird lineages. FITCH topologies estimated from symmetrized delta TmH-C values and subjected to various validation methods (bootstrapping, weighted jackknifing, branch length significance) indicated a fundamental split between hermit (Eutoxeres aquila, Threnetes ruckeri; Phaethornithinae) and nonhermit (Trochilinae) hummingbirds, and provided strong support for six principal nonhermit clades with the following branching order: (1) a predominantly lowland group comprising caribs (Eulampis holosericeus) and relatives (Androdon aequatorialis and Heliothryx barroti) with violet-ears (Colibri coruscans) and relatives (Doryfera ludovicae); (2) an Andean-associated clade of highly polytypic taxa (Eriocnemis, Heliodoxa, and Coeligena); (3) a second endemic Andean clade (Oreotrochilus chimborazo, Aglaiocercus coelestis, and Lesbia victoriae) paired with thorntails (Popelairia conversii); (4) emeralds and relatives (Chlorostilbon mellisugus, Amazilia tzacatl, Thalurania colombica, Orthorhyncus cristatus and Campylopterus villaviscensio); (5) mountain-gems (Lampornis clemenciae and Eugenes fulgens); and (6) tiny bee-like forms (Archilochus colubris, Myrtis fanny, Acestrura mulsant, and Philodice mitchellii). Corresponding analyses on a matrix of unsymmetrized delta values gave similar support for these relationships except that the branching order of the two Andean clades (2, 3 above) was unresolved. In general, subsidiary relationships were consistent and well supported by both matrices, sometimes revealing surprising associations between forms that differ dramatically in plumage and bill morphology. Our results also reveal some

  3. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  4. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.

    Science.gov (United States)

    Li, Ting; Huang, Sheng; Jiang, Wen Zhi; Wright, David; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-01-01

    DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

  5. Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae).

    Science.gov (United States)

    Nishimoto, Yuriko; Ohnishi, Ohmi; Hasegawa, Masami

    2003-04-01

    We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.

  6. Screening and identification of male-specific DNA fragments in common carps Cyprinus carpio using suppression subtractive hybridization.

    Science.gov (United States)

    Chen, J J; Du, Q Y; Yue, Y Y; Dang, B J; Chang, Z J

    2010-08-01

    In this study, a sex subtractive genomic DNA library was constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. Twenty-two clones with distinguishable hybridization signals were selected and sequenced. The specific primers were designed based on the sequence data. Those primers were then used to amplify the sex-specific fragments from the genomic DNA of male and female carp. The amplified fragments from two clones showed specificity to males but not to females, which were named as Ccmf2 [387 base pairs (bp)] and Ccmf3 (183 bp), respectively. The sex-specific pattern was analysed in a total of 40 individuals from three other different C. carpio. stocks and grass carp Ctenopharyngodon idella using Ccmf2 and Ccmf3 as dot-blotting probes. The results revealed that the molecular diversity exists on the Y chromosome of C. carpio. No hybridization signals, however, were detected from individuals of C. idella, suggesting that the two sequences are specific to C. carpio. No significant homologous sequences of Ccmf2 and Ccmf3 were found in GenBank. Therefore, it was interpreted that the results as that Ccmf2 and Ccmf3 are two novel male-specific sequences; and both fragments could be used as markers to rapidly and accurately identify the genetic sex of part of C. carpio. This may provide a very efficient selective tool for practically breeding monosex female populations in aquacultural production.

  7. DETECTION OF STRAND BREAKS OF DNA IN HUMAN EARLY CHORIONIC VILLUS CELLS INDUCED BY DIAGNOSTIC ULTRASOUND USING 32p-LABELED ALU HYBRIDIZATION

    Institute of Scientific and Technical Information of China (English)

    Wang Caifeng; Li Xu; Zhang Yunjing

    2006-01-01

    Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and doublestranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5 × 103 chorionic villus cells and 0.45 ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method, 32P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.

  8. Sensitive optical bio-sensing of p-type WSe2 hybridized with fluorescent dye attached DNA by doping and de-doping effects

    Science.gov (United States)

    Han, Kyu Hyun; Kim, Jun Young; Jo, Seong Gi; Seo, Changwon; Kim, Jeongyong; Joo, Jinsoo

    2017-10-01

    Layered transition metal dichalcogenides, such as MoS2, WSe2 and WS2, are exciting two-dimensional (2D) materials because they possess tunable optical and electrical properties that depend on the number of layers. In this study, the nanoscale photoluminescence (PL) characteristics of the p-type WSe2 monolayer, and WSe2 layers hybridized with the fluorescent dye Cy3 attached to probe-DNA (Cy3/p-DNA), have been investigated as a function of the concentration of Cy3/DNA by using high-resolution laser confocal microscopy. With increasing concentration of Cy3/p-DNA, the measured PL intensity decreases and its peak is red-shifted, suggesting that the WSe2 layer has been p-type doped with Cy3/p-DNA. Then, the PL intensity of the WSe2/Cy3/p-DNA hybrid system increases and the peak is blue-shifted through hybridization with relatively small amounts of target-DNA (t-DNA) (50–100 nM). This effect originates from charge and energy transfer from the Cy3/DNA to the WSe2. For t-DNA detection, our systems using p-type WSe2 have the merit in terms of the increase of PL intensity. The p-type WSe2 monolayers can be a promising nanoscale 2D material for sensitive optical bio-sensing based on the doping and de-doping responses to biomaterials.

  9. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic...

  10. Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata.

    Science.gov (United States)

    Hoareau, T B; Boissin, E

    2010-11-01

    Recent research has shown the usefulness of the Folmer region of the cytochrome oxidase I (COI) as a genetic barcode to assist in species delimitation of echinoderms. However, amplification of COI is often challenging in echinoderms (low success or pseudogenes). We present a method that allows the design of phylum-specific hybrid primers, and use this to develop COI primers for the Echinodermata. We aligned COI sequences from 310 echinoderm species and designed all possible primers along the consensus sequence with two methods (standard degenerate and hybrid). We found much lower degeneracy for hybrid primers (4-fold degeneracy) than for standard degenerate primers (≥48-fold degeneracy). We then designed the most conserved hybrid primers to amplify a >500-bp region within COI. These primers successfully amplified this gene region in all tested taxa (123 species across all echinoderm classes). Sequencing of 30 species among these confirmed both the quality of the sequences (>500 bp, no pseudogenes) and their utility as a DNA barcode. This method should be useful for developing primers for other mitochondrial genes and other phyla. The method will also be of interest for the development of future projects involving both community-based genetic assessments on macroorganisms and biodiversity assessment of environmental samples using high-throughput sequencing.

  11. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

    Science.gov (United States)

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C.

    2014-01-01

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

  12. Evaluation of the Gibbs Free Energy Changes and Melting Temperatures of DNA/DNA Duplexes Using Hybridization Enthalpy Calculated by Molecular Dynamics Simulation.

    Science.gov (United States)

    Lomzov, Alexander A; Vorobjev, Yury N; Pyshnyi, Dmitrii V

    2015-12-10

    A molecular dynamics simulation approach was applied for the prediction of the thermal stability of oligonucleotide duplexes. It was shown that the enthalpy of the DNA/DNA complex formation could be calculated using this approach. We have studied the influence of various simulation parameters on the secondary structure and the hybridization enthalpy value of Dickerson-Drew dodecamer. The optimal simulation parameters for the most reliable prediction of the enthalpy values were determined. The thermodynamic parameters (enthalpy and entropy changes) of a duplex formation were obtained experimentally for 305 oligonucleotides of various lengths and GC-content. The resulting database was studied with molecular dynamics (MD) simulation using the optimized simulation parameters. Gibbs free energy changes and the melting temperatures were evaluated using the experimental correlation between enthalpy and entropy changes of the duplex formation and the enthalpy values calculated by the MD simulation. The average errors in the predictions of enthalpy, the Gibbs free energy change, and the melting temperature of oligonucleotide complexes were 11%, 10%, and 4.4 °C, respectively. We have shown that the molecular dynamics simulation gives a possibility to calculate the thermal stability of native DNA/DNA complexes a priori with an unexpectedly high accuracy.

  13. Interspecies comparative genome hybridization and interspecies representational difference analysis reveal gross DNA differences between humans and great apes.

    Science.gov (United States)

    Toder, R; Xia, Y; Bausch, E

    1998-09-01

    Comparative chromosome G-/R-banding, comparative gene mapping and chromosome painting techniques have demonstrated that only few chromosomal rearrangements occurred during great ape and human evolution. Interspecies comparative genome hybridization (CGH), used here in this study, between human, gorilla and pygmy chimpanzee revealed species-specific regions in all three species. In contrast to the human, a far more complex distribution of species-specific blocks was detected with CGH in gorilla and pygmy chimpanzee. Most of these blocks coincide with already described heterochromatic regions on gorilla and chimpanzee chromosomes. Representational difference analysis (RDA) was used to subtract the complex genome of gorilla against human in order to enrich gorilla-specific DNA sequences. Gorilla-specific clones isolated with this technique revealed a 32-bp repeat unit. These clones were mapped by fluorescence in situ hybridization (FISH) to the telomeric regions of gorilla chromosomes that had been shown by interspecies CGH to contain species-specific sequences.

  14. Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization.

    Science.gov (United States)

    Mills, D; Russell, B W; Hanus, J W

    1997-08-01

    ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

  15. Use of 3,3',5,5' tetramethylbenzidine as new electrochemical indicator of DNA hybridization and its application in genossensor.

    Science.gov (United States)

    Alves-Balvedi, R P; Caetano, L P; Madurro, J M; Brito-Madurro, A G

    2016-11-15

    Electrochemical tools are important biosensor platforms for disease diagnosis, due to their speediness, easiness, low cost and portability. However, for DNA detection, the use of indicators and/or intercalators is necessary to improve electrochemical sensitivity. Currently, ethidium bromide (EthBr) is the cheapest and most used DNA intercalators, but presents carcinogenic and teratogenic properties. Other indicators may be important for DNA photonic detection, and besides being more expensive, they behave similarly to EthBr. This investigation shows for the first time the use of tetramethylbenzidine(TMB) as a new remarkable non-carcinogenic DNA indicator for genosensing purposes, which may be used for nucleic acid detection of microorganisms, based on complementarity of base-pairing between probe and target molecules. The results indicate that TMB can be used as a new electrochemical indicator readily applicable in genosensors, which is able to detect the hybridization of single stranded DNA probe with its complementary target strand. An additional advantage of TMB, beside its non-genotoxicity, is the electrochemical reduction property, which prevents interference of serum components and other oxidative samples in the electrochemical analysis.

  16. Molecular evidence for the hybrid origin of Paulownia Taiwaniana based on RAPD markers and RFLP of chloroplast DNA.

    Science.gov (United States)

    Wang, W Y; Pai, R C; Lai, C C; Lin, T P

    1994-10-01

    Genomic DNA of Paulownia fortunei, P. kawakamii and P. taiwaniana were amplified with 10-base primers of arbitrary sequences using the polymerase chain reaction (PCR). A total of 351 DNA fragments were amplified from 23 primers and of these 265 fragments (75.5%) were polymorphic. Almost all of the PCR-amplified products of P. taiwaniana were shared by either P. fortunei or P. kawakamii, or both, and the number of polymorphic fragments shared by P. taiwaniana and P. fortunei was about equivalent to those shared by P. taiwaniana and P. kawakamii. Restriction fragments of chloroplast DNA (cpDNA) purified from Paulownia species and from reciprocal crosses between P. fortunei and P. kawakamii were analyzed. Restriction enzyme SalI-digested cpDNA showed an identical pattern in both P. kawakamii and P. taiwaniana. These results further support the hypothesis that P. taiwaniana is the natural hybrid between P. fortunei and P. kawakamii and that the maternal parent of P. taiwaniana is P. kawakamii.

  17. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae.

    Science.gov (United States)

    Jeon, Junhyun; Choi, Jaeyoung; Lee, Gir-Won; Park, Sook-Young; Huh, Aram; Dean, Ralph A; Lee, Yong-Hwan

    2015-02-24

    DNA methylation is an important epigenetic modification that regulates development of plants and mammals. To investigate the roles of DNA methylation in fungal development, we profiled genome-wide methylation patterns at single-nucleotide resolution during vegetative growth, asexual reproduction, and infection-related morphogenesis in a model plant pathogenic fungus, Magnaporthe oryzae. We found that DNA methylation occurs in and around genes as well as transposable elements and undergoes global reprogramming during fungal development. Such reprogramming of DNA methylation suggests that it may have acquired new roles other than controlling the proliferation of TEs. Genetic analysis of DNA methyltransferase deletion mutants also indicated that proper reprogramming in methylomes is required for asexual reproduction in the fungus. Furthermore, RNA-seq analysis showed that DNA methylation is associated with transcriptional silencing of transposable elements and transcript abundance of genes in context-dependent manner, reinforcing the role of DNA methylation as a genome defense mechanism. This comprehensive approach suggests that DNA methylation in fungi can be a dynamic epigenetic entity contributing to fungal development and genome defense. Furthermore, our DNA methylomes provide a foundation for future studies exploring this key epigenetic modification in fungal development and pathogenesis.

  18. 致病性小肠结肠耶氏菌DNA多态性及其毒力表达%DNA POLYMORPHISM AND PATHOGENIC EXPRESSION OF YERSINIA ENTEROCOLITICA

    Institute of Scientific and Technical Information of China (English)

    王军; 王红旗; 李跃旗; 施红; 王全立; 刘耀清; 马立仁

    2001-01-01

    目的探讨致病性小肠结肠耶氏菌(简称:耶氏菌)DNA特征及其表达。方法用聚合酶链反应(polymeraseChain Reaction;PCR)、DNA序列分析、随机扩增多态性DNA(Randomly Amplified Polymorphic DNA;RAPD)检测耶氏茵致病基因(adherent invasionlocus;ail),并用自凝试验、刚果红试验检测其毒力表达。结果PCR和DNA序列分析证实致病性耶氏菌O:3、O:9、O:5,27血清型含有ail致病基因,而非致病型O:4,33、O:7,8血清型不含有ail基因。此外,O:22血清型也含有ail基因与文献报告ai1核苷酸序列同源性为88.3%。O:3、O:9、O:5,27致病血清型DNA指纹图谱相类似,O:22血清型有部分DNA片段与0:3血清型相似,而非致病O:4,33血清型DNA指纹图谱完全不同。毒力试验显示致病性耶氏茵全部阳性,3株O:22血清型均有少数菌落刚果试验阳性。结论致病性耶氏菌含有ail致病基因,DNA指纹图谱相似,但表现为多态性。O:22血清型含有ail基因,DNA指纹图谱部分与O:3血清型相似,少数菌落毒力试验呈阳性反应,该血清型可能存在潜在致病性。%Aim To study DNA characteristics and pathogenic expression of Yersinis enterocolitica. Method Polymerase chain reaction (PCR), analysis of DNA sequence, randomly amplified polymorphic DNA (RAPD) were used to investigate the pathogenic gene of adherent invasion locus (ail)and autoagglutination test and Congo red to observe the pathogenic expression of Yersinia enterocolitica. Results The Pathogenic Yersinia enteracolitica of serotyps O: 3、O: 9 and O:5, 27 were confirmed containing ail gene and the nonpathogenic of O:4, 33 and O: 7, 8 containing no one with PCR and analysis of DNA sequence. In addition,serotype O: 22 also contained ail gene which its nucleic acid sequence had homology of 88.3 % compared with the reported. There were similar DNA fingerprints among serotype O: 3 、 O: 9 and O: 5, 27 and

  19. MELAS-like encephalomyopathy caused by a new pathogenic mutation in the mitochondrial DNA encoded cytochrome c oxidase subunit I.

    Science.gov (United States)

    Lamperti, Costanza; Diodato, Daria; Lamantea, Eleonora; Carrara, Franco; Ghezzi, Daniele; Mereghetti, Paolo; Rizzi, Romana; Zeviani, Massimo

    2012-11-01

    We report a 35-year-old woman presenting a stroke-like episode with transitory aphasia followed by generalized tonic-clonic seizures. She had severe hearing loss and suffered from frequent episodes of migraine. Although a brain MRI disclosed a T2-hyperintense lesion in the left parietal lobe, she had hardly any long-term sequela. Exercise intolerance, myalgias and limb-girdle muscle weakness indicated a slowly progressive myopathy. Extra-neurological features included short stature, and secondary amenorrhea with low gonadotropin levels, indicating secondary hypogonadism. However, she had three mutation-free, healthy children by ovarian stimulation. A muscle biopsy showed ragged-red, cytochrome c oxidase-negative fibers, and an isolated defect of cytochrome c oxidase activity in muscle mitochondria. Sequence analysis of muscle mtDNA revealed a previously unreported heteroplasmic m.6597C>A transversion in the MTCOI gene, encoding subunit I of cytochrome c oxidase, corresponding to p.Q232K aminoacid change. Analysis on transmitochondrial cybrids demonstrated that the mutation is indeed associated with COX deficiency, i.e. pathogenic.

  20. Comparison of different inoculating methods to evaluate the pathogenicity and virulence of Aspergillus niger on two maize hybrids

    Science.gov (United States)

    A two-year field study was conducted to determine the effects of inoculation techniques on the aggressiveness of Aspergillus niger kernel infection in A. flavus resistant and susceptible maize hybrids. Ears were inoculated with the silk-channel, side-needle, and spray techniques 7 days after midsilk...

  1. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Directory of Open Access Journals (Sweden)

    Marijn T J van Loenhout

    Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  2. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Science.gov (United States)

    van Loenhout, Marijn T J; De Vlaminck, Iwijn; Flebus, Benedetta; den Blanken, Johan F; Zweifel, Ludovit P; Hooning, Koen M; Kerssemakers, Jacob W J; Dekker, Cees

    2013-01-01

    The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  3. A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH)

    Institute of Scientific and Technical Information of China (English)

    Ke BI; James P.BOGART; Jinzhong FU

    2009-01-01

    The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution [Current Zoology 55(2):145-149,2009].

  4. A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH

    Directory of Open Access Journals (Sweden)

    Jinzhong FU

    2009-04-01

    Full Text Available The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA. Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution.

  5. Surface-enhanced localized surface plasmon resonance biosensing of avian influenza DNA hybridization using subwavelength metallic nanoarrays

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Shin Ae; Jang, Sung Min; Kim, Sung June [School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-742 (Korea, Republic of); Byun, Kyung Min [Department of Biomedical Engineering, Kyung Hee University, Yongin 446-701 (Korea, Republic of); Kim, Kyujung; Kim, Donghyun [Program of Nanomedical Science and Technology, Yonsei University, Seoul 120-749 (Korea, Republic of); Ma, Kyungjae; Oh, Youngjin [School of Electrical and Electronic Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Sung Guk [College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 (United States); Shuler, Michael L, E-mail: kmbyun@khu.ac.kr [Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853 (United States)

    2010-09-03

    We demonstrated enhanced localized surface plasmon resonance (SPR) biosensing based on subwavelength gold nanoarrays built on a thin gold film. Arrays of nanogratings (1D) and nanoholes (2D) with a period of 200 nm were fabricated by electron-beam lithography and used for the detection of avian influenza DNA hybridization. Experimental results showed that both nanoarrays provided significant sensitivity improvement and, especially, 1D nanogratings exhibited higher SPR signal amplification compared with 2D nanohole arrays. The sensitivity enhancement is associated with changes in surface-limited reaction area and strong interactions between bound molecules and localized plasmon fields. Our approach is expected to improve both the sensitivity and sensing resolution and can be applicable to label-free detection of DNA without amplification by polymerase chain reaction.

  6. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

    Science.gov (United States)

    Lewis, J B; Atkins, J F; Anderson, C W; Baum, P R; Gesteland, R F

    1975-04-01

    Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R-EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides. From the known order of the R-EcoRI fragments, the following partial map is deduced: (III, IIIa, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

  7. Evolutionary analysis of the CACTA DNA-transposon Caspar across wheat species using sequence comparison and in situ hybridization.

    Science.gov (United States)

    Sergeeva, Ekaterina M; Salina, Elena A; Adonina, Irina G; Chalhoub, Boulos

    2010-07-01

    Mobile elements constitute a considerable part of the eukaryotic genome. This work is focused on the distribution and evolution of DNA-transposons in the genomes of diploid and allopolyploid Triticeae species and their role in the formation of functionally important chromosomal subtelomeric regions. The Caspar family is among the most abundant of CACTA DNA-transposons in Triticeae. To study the evolution of Caspar-like elements in Triticeae genomes, we analyzed their sequences and distribution in chromosomes by in situ hybridization. In total, 46 Caspar-like elements from the wheat and barley Caspar, Clifford, and Donald families were analyzed after being extracted from databases using the transposase consensus sequence. Sequence alignment and subsequent phylogenetic analyses revealed that the transposase DNA sequences formed three major distinct groups: (1) Clifford, (2) Caspar_Triticinae, and (3) Caspar_Hordeinae. Additionally, in situ hybridization demonstrated that Caspar_Triticinae transposons are predominantly compartmentalized in the subtelomeric chromosomal regions of wheat and its progenitors. Analysis of data suggested that compartmentalization in the subtelomeric chromosomal region was a characteristic feature of all the main groups of Caspar-like elements. Furthermore, a dot plot analysis of the terminal repeats demonstrated that the divergence of these repeats strictly correlated with the divergence of Caspar coding sequences. A clear distinction in the Caspar DNA sequences among the species Triticum/Aegilops (Caspar_Triticinae), Hordeum (Caspar_Hordeinae), and different distributions in individual hexaploid wheat genomes (A/B and D) suggest an independent proliferation of these elements in wheat (or its progenitors) and barley genomes. Thus, Caspar-like transposons can significantly contribute to the formation and differentiation of subtelomeric regions in Triticeae species.

  8. Polydopamine-immobilized polypropylene microfuge tube as a pH-responsive platform for capture/release of DNA from foodborne pathogens.

    Science.gov (United States)

    Zhong, Zitao; Yao, Xin; Gao, Xiaomei; Jia, Li

    2017-10-01

    A rapid, convenient and efficient DNA extraction method with no need for toxic agents and centrifugation was reported. A polypropylene microfuge (MF) tube was used as the substrate to immobilize polydopamine (PDA). The prepared PDA-coated MF (PDA@MF) tube was used as a pH-responsive platform for rapid extraction of DNA based on pH-induced charge switch of amino and phenolic hydroxyl groups in PDA coating. The extraction procedure is simple and can be finished in 25 min. The PDA@MF tube was applied for extraction of genomic DNA from foodborne pathogens in milk. The extracted DNA was directly used as template for PCR amplification. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi.

    Science.gov (United States)

    Zhang, Yueping; Qu, Huaxiang; Zhao, Ping; Tang, Yongping; Zhou, Jingsong; Luo, Shaochun; Yin, Yuling; Chen, Guangyu

    2017-07-20

    The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1 × 10(6) conidia. Our results indicated that 25 °C, acetosyringone concentration of 150 μmol/L, and 72 h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2 × PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

  10. Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

    Science.gov (United States)

    Gao, Ran; Lu, Dan-Feng; Cheng, Jin; Jiang, Yi; Jiang, Lan; Xu, Jian-Dong; Qi, Zhi-Mei

    2016-12-15

    An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity.

  11. Fabrication of Silicon Nitride Ion Sensitive Field-Effect Transistor for pH Measurement and DNA Immobilization/Hybridization

    Directory of Open Access Journals (Sweden)

    U. Hashim

    2013-01-01

    Full Text Available The fabrication of ion sensitive field-effect transistor (ISFET using silicon nitride (Si3N4 as the sensing membrane for pH measurement and DNA is reported. For the pH measurement, the Ag/AgCl electrode was used as the reference electrode, and different pH values of buffer solution were used in the ISFET analysis. The ISFET device was tested with pH buffer solutions of pH2, pH3, pH7, pH8, and pH9. The results show that the IV characteristic of ISFET devices is directly proportional and the device’s sensitivity was 43.13 mV/pH. The ISFET is modified chemically to allow the integration with biological element to form a biologically active field-effect transistor (BIOFET. It was found that the DNA immobilization activities which occurred on the sensing membrane caused the drain current to drop due to the negatively charged backbones of the DNA probes repelled electrons from accumulating at the conducting channel. The drain current was further decreased when the DNA hybridization took place.

  12. DNA生物传感器快速检测病原微生物的临床实验研究%Experimental studtes on DNA biosensors for rapid detection of pathogenic microorganism

    Institute of Scientific and Technical Information of China (English)

    张诒亮; 任玉琰

    2013-01-01

    目的:对DNA生物传感器在病原微生物的快速检测方面的临床实验效果进行分析探讨。方法采用SPR传感器对甲型流感病毒进行微生物快速检测。结果 SPR传感器在与流感病毒的核酸进行杂交后,能够灵敏、准确地将病毒的折射率反映出来,且效率和敏感性都比较高。结论 DNA生物传感器相较于传统检测方法更具优势,具有高通量、高敏感、高效率以及低成本等优点,值得推广应用。%Objective To evaluate the clinical effect of DNA biosensor for rapid detection of pathogenic microorganisms. Methods Using the SPR sensor for rapid detection of microorganisms on influenza a virus. Results SPR sensor in nucleic acid hybridization with influenza virus, the virus can sensitively, accurately reflected the refractive index, and the efficiency and sensitivity were high. Conclusion The traditional method of detecting DNA biosensor compared with more advantages, the advantages of high throughput, high sensitive, high efficiency and low cost, and is worthy of popularization and application.

  13. Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization.

    Directory of Open Access Journals (Sweden)

    Renhai Peng

    Full Text Available Fluorescence in situ hybridization (FISH has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH

  14. A new hybrid bacteriocin, Ent35–MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

    Science.gov (United States)

    Acuña, Leonardo; Picariello, Gianluca; Sesma, Fernando; Morero, Roberto D.; Bellomio, Augusto

    2012-01-01

    Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries. PMID:23650575

  15. Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Ma, Xingyi; Cao, Cuong

    2011-01-01

    In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target...... in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which...

  16. Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray.

    Directory of Open Access Journals (Sweden)

    Taro Ueno

    Full Text Available MicroRNAs (miRNAs can be used as biomarkers for cancer and other human diseases; therefore, high-throughput and reliable miRNA-quantification methods are required to exploit these markers for diagnostic testing. In this report, we describe the construction of a platform for miRNA-quantification using ligase-assisted sandwich hybridization (LASH without miRNA-labeling. T4 DNA ligase was used to compensate for the low affinity between miRNAs and two short complementary DNA probes, and it improved the hybridization yield ∼50,000 times. The LASH assay enabled synthesized miR-143 to be quantified at concentrations ranging from 30 fM to 30 pM. The LASH assay could also quantify endogenous miR-143 released from cultured cells as well as some miRNAs in total RNAs derived from blood. Furthermore, multi-color detection enabled us to distinguish between the highly homologous miR-141 and miR-200a. This simple label-free quantification technique is an easy-to-use approach that can be applied to disease diagnosis.

  17. 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.

  18. Does hybridization increase evolutionary rate? Data from the 28S-rDNA D8 domain in echinoderms.

    Science.gov (United States)

    Chenuil, Anne; Egea, Emilie; Rocher, Caroline; Touzet, Hélène; Féral, Jean-Pierre

    2008-11-01

    The divergent domain D8 of the large ribosomal RNA is very variable and extended in vertebrates compared to other eukaryotes. We provide data from 31 species of echinoderms and present the first comparative analysis of the D8 in nonvertebrate deuterostomes. In addition, we obtained 16S mitochondrial DNA sequences for the sea urchin taxa and analyzed single-strand conformation polymorphism (SSCP) of D8 in several populations within the species complex Echinocardium cordatum. A common secondary structure supported by compensatory substitutions and indels is inferred for echinoderms. Variation mostly arises at the tip of the longest stem (D8a), and the most variable taxa also display the longest and most stable D8. The most stable variants are the only ones displaying bulges in the terminal part of the stem, suggesting that selection, rather than maximizing stability of the D8 secondary structure, maintains it in a given range. Striking variation in D8 evolutionary rates was evidenced among sea urchins, by comparison with both 16S mitochondrial DNA and paleontological data. In Echinocardium cordatum and Strongylocentrotus pallidus and S. droebachiensis, belonging to very distant genera, the increase in D8 evolutionary rate is extreme. Their highly stable D8 secondary structures rule out the possibility of pseudogenes. These taxa are the only ones in which interspecific hybridization was reported. We discuss how evolutionary rates may be affected in nuclear relative to mitochondrial genes after hybridization, by selective or mutational processes such as gene silencing and concerted evolution.

  19. 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.

  20. Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

    Science.gov (United States)

    Lewis, J B; Anderson, C W; Atkins, J F

    1977-09-01

    RNA isolated from the cytoplasm of human cells at late times after infection by adenovirus type 2 (Ad2) has been fractionated by hybridization to fragments of Ad2 DNA which were produced by digestion with the restriction endonucleases Hpa I, Eco RI, Bam HI and Hind III. Cell-free translation of these partially purified mRNAs indicates that the genes for the late Ad2 proteins lie within the following intervals on the conventional Ad2 map: 15K (4.4-17.0 map units), IX and IVa2 (7.5-17.0), IIIa (29.1-40.9), III and V (29.1-57.0), pVIII (40.9-57.0), pVI and II (40.9-70.7), 100K (59.0-83.4), pVIII (70.7-83.4) and IV (85.0-100). In addition to the primary hybridization of the late Ad2 mRNAs to the regions indicated above, most late Ad2 mRNAs (except those for 15K, IX and IVa2) exhibited some hybridization to a secondary site between 17.0 and 29.1 map units.

  1. Development of Convergence Nanoparticles (Phase II): Detection and Therapeutics of Pathogen Targets by Using Multi-Mode Hybrid Nanoparticle Probe

    Science.gov (United States)

    2010-04-05

    using heat generation effect of magnetic component. 15. SUBJECT TERMS Bio -applications, Nanotechnology 16. SECURITY CLASSIFICATION OF: 17...for the next generation biomedical sensing techniques, which will be very useful for anti-pathogenic and anti- bio /chemical warfares as well as normal...components such as magnetic, bio -active, optical, radioactive, and heat-generating materials into a single nanosystem via molecular linkers, it was

  2. Proposal of Xanthomonas translucens pv. pistaciae pv. nov., pathogenic to pistachio (Pistacia vera).

    Science.gov (United States)

    Giblot-Ducray, Danièle; Marefat, Alireza; Gillings, Michael R; Parkinson, Neil M; Bowman, John P; Ophel-Keller, Kathy; Taylor, Cathy; Facelli, Evelina; Scott, Eileen S

    2009-12-01

    Strains of Xanthomonas translucens have caused dieback in the Australian pistachio industry for the last 15 years. Such pathogenicity to a dicotyledonous woody host contrasts with that of other pathovars of X. translucens, which are characterized by their pathogenicity to monocotyledonous plant families. Further investigations, using DNA-DNA hybridization, gyrB gene sequencing and integron screening, were conducted to confirm the taxonomic status of the X. translucens pathogenic to pistachio. DNA-DNA hybridization provided a clear classification, at the species level, of the pistachio pathogen as a X. translucens. In the gyrB-based phylogeny, strains of the pistachio pathogen clustered among the X. translucens pathovars as two distinct lineages. Integron screening revealed that the cassette arrays of strains of the pistachio pathogen were different from those of other Xanthomonas species, and again distinguished two groups. Together with previously reported pathogenicity data, these results confirm that the pistachio pathogen is a new pathovar of X. translucens and allow hypotheses about its origin. The proposed name is Xanthomonas translucens pv. pistaciae pv. nov.

  3. Hybridization State Detection of DNA-Functionalized Gold Nanoparticles Using Hyperspectral Imaging

    Directory of Open Access Journals (Sweden)

    Richard C. Murdock

    2017-01-01

    Full Text Available Hyperspectral imaging has the unique ability of capturing spectral data for multiple wavelengths at each pixel in an image. This gives the ability to distinguish, with certainty, different nanomaterials and/or distinguish nanomaterials from biological materials. In this study, 4 nm and 13 nm gold nanoparticles (Au NPs were synthesized, functionalized with complimentary oligonucleotides, and hybridized to form large networks of NPs. Scattering spectra were collected from each sample (unfunctionalized, functionalized, and hybridized and evaluated. The spectra showed unique peaks for each size of Au NP sample and also exhibited narrowing and intensifying of the spectra as the NPs were functionalized and then subsequently hybridized. These spectra are different from normal aggregation effects where the LSPR and reflected spectrum broaden and are red-shifted. Rather, this appears to be dependent on the ability to control the interparticle distance through oligonucleotide length, which is also investigated through the incorporation of a poly-A spacer. Also, hybridized Au NPs were exposed to cells with no adverse effects and retained their unique spectral signatures. With the ability to distinguish between hybridization states at nearly individual NP levels, this could provide a new method of tracking the intracellular actions of nanomaterials as well as extracellular biosensing applications.

  4. Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling in Magnaporthe oryzae

    Science.gov (United States)

    Wáng, Ying; Tan, Qi; Gao, Ying Nv; Li, Yan

    2017-01-01

    High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity in Magnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity in M. oryzae identified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.

  5. 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.

  6. Implementation of hybrid clustering based on partitioning around medoids algorithm and divisive analysis on human Papillomavirus DNA

    Science.gov (United States)

    Arimbi, Mentari Dian; Bustamam, Alhadi; Lestari, Dian

    2017-03-01

    Data clustering can be executed through partition or hierarchical method for many types of data including DNA sequences. Both clustering methods can be combined by processing partition algorithm in the first level and hierarchical in the second level, called hybrid clustering. In the partition phase some popular methods such as PAM, K-means, or Fuzzy c-means methods could be applied. In this study we selected partitioning around medoids (PAM) in our partition stage. Furthermore, following the partition algorithm, in hierarchical stage we applied divisive analysis algorithm (DIANA) in order to have more specific clusters and sub clusters structures. The number of main clusters is determined using Davies Bouldin Index (DBI) value. We choose the optimal number of clusters if the results minimize the DBI value. In this work, we conduct the clustering on 1252 HPV DNA sequences data from GenBank. The characteristic extraction is initially performed, followed by normalizing and genetic distance calculation using Euclidean distance. In our implementation, we used the hybrid PAM and DIANA using the R open source programming tool. In our results, we obtained 3 main clusters with average DBI value is 0.979, using PAM in the first stage. After executing DIANA in the second stage, we obtained 4 sub clusters for Cluster-1, 9 sub clusters for Cluster-2 and 2 sub clusters in Cluster-3, with the BDI value 0.972, 0.771, and 0.768 for each main cluster respectively. Since the second stage produce lower DBI value compare to the DBI value in the first stage, we conclude that this hybrid approach can improve the accuracy of our clustering re