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Sample records for cytometry-based dna hybridization

  1. Flow-cytometry-based DNA hybidization and polymorphism analysis

    NASA Astrophysics Data System (ADS)

    Cai, Hong; Kommander, Kristina; White, P. S.; Nolan, John P.

    1998-05-01

    Functional analysis of the human 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. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our 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 advantages 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.

  2. A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells.

    PubMed

    Rouget, Raphael; Auclair, Yannick; Loignon, Martin; Affar, El Bachir; Drobetsky, Elliot A

    2008-02-29

    In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

  3. DNA-based hybrid catalysis.

    PubMed

    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.

  4. Detecting hybridization using ancient DNA.

    PubMed

    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.

  5. Electrokinetic acceleration of DNA hybridization in microsystems.

    PubMed

    Lei, Kin Fong; Wang, Yun-Hsiang; Chen, Huai-Yi; Sun, Jia-Hong; Cheng, Ji-Yen

    2015-06-01

    In this work, electrokinetic acceleration of DNA hybridization was investigated by different combinations of frequencies and amplitudes of actuating electric signals. Because the frequencies from low to high can induce different kinds of electrokinetic forces, i.e., electroosmotic to electrothermal forces, this work provides an in-depth investigation of electrokinetic enhanced hybridization. Concentric circular Cr/Au microelectrodes of 350 µm in diameter were fabricated on a glass substrate and probe DNA was immobilized on the electrode surface. Target DNA labeled with fluorescent dyes suspending in solution was then applied to the electrode. Different electrokinetic forces were induced by the application of different electric signals to the circular microelectrodes. Local microfluidic vortexes were generated to increase the collision efficiency between the target DNA suspending in solution and probe DNA immobilized on the electrode surface. DNA hybridization on the electrode surface could be accelerated by the electrokinetic forces. The level of hybridization was represented by the fluorescent signal intensity ratio. Results revealed that such 5-min dynamic hybridization increased 4.5 fold of signal intensity ratio as compared to a 1-h static hybridization. Moreover, dynamic hybridization was found to have better differentiation ability between specific and non-specific target DNA. This study provides a strategy to accelerate DNA hybridization in microsystems. PMID:25863384

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

    PubMed

    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.

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

    PubMed

    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. PMID:25102381

  8. Detection of pseudorabies virus by DNA hybridization

    SciTech Connect

    McFarlane, R.G.

    1985-01-01

    A DNA hybridization technique was developed in order to detect the presence of pseudorabies virus (PRV) deoxyribonucleic acid (DNA) in swine tissue. Seven, /sup 32/P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV-DNA had been inserted. Under optimal hybridization conditions, the minimum detection level of PRV-DNA was 10/sup -11/ g, which is equivalent to 1 copy of the PRV genome/80 host cells. PRV-DNA was detected in the DNA extracted from the tissues of 10 out of 11 swine previously shown to harbor infective virus. Furthermore, PRV-DNA was present in all four seropositive swine that had recovered from pseudorabies, where no infective virus or viral products were detected at necropsy. The PRV-DNA was present in either the anterior cerebral cortex in 2 swine, or the medulla oblongata and trigeminal ganglion in 1 swine. This perhaps indicates the portal of entry of the virus into the central nervous system. This DNA hybridization assay, which utilizes restriction fragments, may be useful for studying the dynamics and molecular biologic properties of the latency of pseudorabies virus in swine.

  9. Control of DNA hybridization with photocleavable adducts.

    PubMed

    Ghosn, Bilal; Haselton, Frederick R; Gee, Kyle R; Monroe, W Todd

    2005-01-01

    Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.

  10. Hybridization and Selective Release of DNA Microarrays

    SciTech Connect

    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 to

  11. Characterization of denaturation and renaturation of DNA for DNA hybridization

    PubMed Central

    Wang, Xiaofang; Lim, Hyun Jeong; Son, Ahjeong

    2014-01-01

    Objectives The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. Methods A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization. PMID:25234413

  12. Automated DNA electrophoresis, hybridization and detection

    SciTech Connect

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-05-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; /sup 32/P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing.

  13. DNA-DNA hybridization evidence of the rapid rate of muroid rodent DNA evolution.

    PubMed

    Catzeflis, F M; Sheldon, F H; Ahlquist, J E; Sibley, C G

    1987-05-01

    Single-copy nuclear DNAs (scnDNAs) of eight species of arvicoline and six species of murine rodents were compared using DNA-DNA hybridization. The branching pattern derived from the DNA comparisons is congruent with the fossil evidence and supported by comparative biochemical, chromosomal, and morphological studies. The recently improved fossil record for these lineages provides seven approximate divergence dates, which were used to calibrate the DNA-hybridization data. The average rate of scnDNA divergence was estimated as 2.5%/Myr. This is approximately 10 times the rate in the hominoid primates. These results agree with previous reports of accelerated DNA evolution in muroid rodents and extend the DNA-DNA hybridization data set of Brownell.

  14. Molecular Mechanisms in Morpholino-DNA Surface Hybridization

    PubMed Central

    Gong, Ping; Wang, Kang; Liu, Yatao; Shepard, Kenneth

    2010-01-01

    Synthetic nucleic acid mimics provide opportunity for redesigning the specificity and affinity of hybridization with natural DNA or RNA. Such redesign is of great interest for diagnostic applications where it can enhance the desired signal against a background of competing interactions. This report compares hybridization of DNA analyte strands with morpholinos (MOs), which are uncharged nucleic acid mimics, to the corresponding DNA-DNA case in solution and on surfaces. In solution, MO-DNA hybridization is found to be independent of counterion concentration, in contrast to DNA-DNA hybridization. On surfaces, when immobilized MO or DNA “probe” strands hybridize with complementary DNA “targets” from solution, both the MO-DNA and DNA-DNA processes depend on ionic strength but exhibit qualitatively different behaviors. At lower ionic strengths, MO-DNA surface hybridization exhibits hallmarks of kinetic limitations when separation between hybridized probe sites becomes comparable to target dimensions, whereas extents of DNA-DNA surface hybridization are instead consistent with limits imposed by buildup of surface (Donnan) potential. The two processes also fundamentally differ at high ionic strength, under conditions when electrostatic effects are weak. Here, variations in probe coverage have a much diminished impact on MO-DNA than on DNA-DNA hybridization for similarly crowded surface conditions. These various observations agree with a structural model of MO monolayers in which MO-DNA duplexes segregate to the buffer interface while unhybridized probes localize near the solid support. A general perspective is presented on using uncharged DNA analogues, which also include compounds such as peptide nucleic acids (PNA), in surface hybridization applications. PMID:20572663

  15. Control of DNA hybridization by photoswitchable molecular glue.

    PubMed

    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.

  16. DNA Hybridization: Nonradioactive Labeling Now Available for the Laboratory.

    ERIC Educational Resources Information Center

    Freeman, Lenore Gardner

    1984-01-01

    The advantages of DNA hybridization procedures for classroom and clinical use can now be realized by the recent development of nonradioactive DNA labeling/detection procedures. These methods (which are described) can replace the use of isotopes in standard DNA hybridization procedures. (JN)

  17. Capture Hybridization Analysis of DNA Targets.

    PubMed

    Sexton, Alec N; Machyna, Martin; Simon, Matthew D

    2016-01-01

    There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells. PMID:27659977

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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. PMID:27533365

  1. Detection of DNA hybridizations using solid-state nanopores

    NASA Astrophysics Data System (ADS)

    Balagurusamy, Venkat S. K.; Weinger, Paul; Ling, Xinsheng Sean

    2010-08-01

    We report an experimental study of using DNA translocation through solid-state nanopores to detect the sequential arrangement of two double-stranded 12-mer hybridization segments on a single-stranded DNA molecule. The sample DNA is a trimer molecule formed by hybridizing three single-stranded oligonucleotides. A polystyrene bead is attached to the end of the trimer DNA, providing a mechanism in slowing down the translocation and suppressing the thermal diffusion, thereby allowing the detection of short features of DNA by standard patch-clamp electronics. The electrical signature of the translocation of a trimer molecule through a nanopore has been identified successfully in the temporal traces of ionic current. The results reported here represent the first successful attempt in using a solid-state nanopore as an ionic scanning device in resolving individual hybridization segments (or 'probes') on a DNA molecule.

  2. Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization

    SciTech Connect

    Datta, A.R.; Wentz, B.A.; Hill, W.E.

    1987-09-01

    A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.

  3. DNA/DNA in situ hybridization with enzyme linked probes

    SciTech Connect

    Grillo, S.; Mosher, M.; Charles, P.; Henry, S.; Taub, F.

    1987-05-01

    A non-radioactive in situ nucleic acid hybridization method which requires no antibodies, haptens, avidin or biotin intermediateries is presented. Horseradish peroxidase (HRP) labeled nucleic acid probes are hybridized in situ for 2 hours or less, followed by brief washing of hybridized cells and the direct detection of in situ hybrids with diaminobenzidine (DAB). Application of this method to the detection of Human Papilloma Virus (HPV) in human cells is shown.

  4. Dynamic Modulation of DNA Hybridization Using Allosteric DNA Tetrahedral Nanostructures.

    PubMed

    Song, Ping; Li, Min; Shen, Juwen; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Wang, Lihua; Shi, Jiye; Song, Shiping; Wang, Lianhui; Fan, Chunhai; Zuo, Xiaolei

    2016-08-16

    The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes. PMID:27435955

  5. Building a Phylogenetic Tree of the Human and Ape Superfamily Using DNA-DNA Hybridization Data

    ERIC Educational Resources Information Center

    Maier, Caroline Alexander

    2004-01-01

    The study describes the process of DNA-DNA hybridization and the history of its use by Sibley and Alquist in simple, straightforward, and interesting language that students easily understand to create their own phylogenetic tree of the hominoid superfamily. They calibrate the DNA clock and use it to estimate the divergence dates of the various…

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

    PubMed

    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.

  7. Cytogenetic analysis from DNA by comparative genomic hybridization.

    PubMed

    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.

  8. Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens.

    PubMed Central

    Farmer, J J; Jorgensen, J H; Grimont, P A; Akhurst, R J; Poinar, G O; Ageron, E; Pierce, G V; Smith, J A; Carter, G P; Wilson, K L

    1989-01-01

    An unusual isolate from a human leg wound was identified as Xenorhabdus luminescens. This finding led to the discovery or isolation of four additional strains, two from blood and two from wounds. Three of the five strains were from patients in San Antonio, Tex. Three strains were studied by DNA-DNA hybridization (S1 nuclease-trichloroacetic acid method) and were 77 to 100% related to each other, 34% related to the type strain of X. luminescens, 35 to 40% related to three of Grimont's other DNA hybridization groups of X. luminescens, and 9% related to the type strain of Xenorhabdus nematophilus. The new group of five strains was designated X. luminescens DNA hybridization group 5. All five strains were very inactive biochemically and fermented only D-glucose and D-mannose. The key reactions for recognizing this new organism are yellow pigment production, negative test for nitrate reduction to nitrite, weak bioluminescence (10 to 15 min of dark adaptation is required to see the weak light produced), and a unique hemolytic reaction on sheep blood agar plates incubated at 25 degrees C. Two case histories of strains from wounds are given; these suggest that X. luminescens DNA hybridization group 5 may be a new bacterial agent that causes wound infections. The two cases of wound infection, along with the two blood isolates, suggest that the new organism is clinically significant. Images PMID:2768446

  9. Kinetic mechanisms in morpholino-DNA surface hybridization.

    PubMed

    Liu, Yatao; Irving, Damion; Qiao, Wanqiong; Ge, Dongbiao; Levicky, Rastislav

    2011-08-01

    Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes. PMID:21699181

  10. Kinetic Mechanisms in Morpholino-DNA Surface Hybridization

    PubMed Central

    Liu, Yatao; Irving, Damion; Qiao, Wanqiong; Ge, Dongbiao

    2011-01-01

    Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or “probe”, species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e. duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes, and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes. PMID:21699181

  11. Role of DNA methylation in hybrid vigor in Arabidopsis thaliana

    PubMed Central

    Kawanabe, Takahiro; Ishikura, Sonoko; Miyaji, Naomi; Sasaki, Taku; Wu, Li Min; Itabashi, Etsuko; Takada, Satoko; Shimizu, Motoki; Takasaki-Yasuda, Takeshi; Osabe, Kenji; Peacock, W. James; Dennis, Elizabeth S.; Fujimoto, Ryo

    2016-01-01

    Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis. PMID:27791039

  12. ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

    NASA Astrophysics Data System (ADS)

    Birrento, Monica L.; Bryan, Tracy M.; Samosorn, Siritron; Beck, Jennifer L.

    2015-07-01

    Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine ( 1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of ( 1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand ( 1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

  13. Coarse-grained DNA modeling: Hybridization and ionic effects

    NASA Astrophysics Data System (ADS)

    Hinckley, Daniel M.

    Deoxyribonucleic acid (DNA) is a biopolymer of enormous significance in living systems. The utility of DNA in such systems is derived from the programmable nature of DNA and its unique mechanical properties. Recently, material scientists have harnessed these properties in order to create systems that spontaneous self-assemble on the nanoscale. Both biologists and material scientists are hindered by an incomplete understanding of the physical interactions that together govern DNA's behavior. Computer simulations, especially those at the coarse-grained (CG) level, can potentially complete this understanding by resolving details indiscernible with current experimental techniques. In this thesis, we advance the state-of-the-art of DNA CG simulations by first reviewing the relevant theory and the evolution of CG DNA models since their inception. Then we present 3SPN.2, an improved CG model for DNA that should provide new insights into biological and nanotechnological systems which incorporate DNA. We perform forward flux sampling simulations in order to examine the effect of sequence, oligomer length, and ionic strength on DNA oligomer hybridization. Due to the limitations inherent in continuum treatments of electrostatic interactions in biological systems, we generate a CG model of biological ions for use with 3SPN.2 and other CG models. Lastly, we illustrate the potential of 3SPN.2 and CG ions by using the models in simulations of viral capsid packaging experiments. The models and results described in this thesis will be useful in future modeling efforts that seek to identify the fundamental physics that govern behavior such as nucleosome positioning, DNA hybridization, and DNA nanoassembly.

  14. Ten-atom silver cluster signaling and tempering DNA hybridization.

    PubMed

    Petty, Jeffrey T; Sergev, Orlin O; Kantor, Andrew G; Rankine, Ian J; Ganguly, Mainak; David, Frederic D; Wheeler, Sandra K; Wheeler, John F

    2015-05-19

    Silver clusters with ∼10 atoms are molecules, and specific species develop within DNA strands. These molecular metals have sparsely organized electronic states with distinctive visible and near-infrared spectra that vary with cluster size, oxidation, and shape. These small molecules also act as DNA adducts and coordinate with their DNA hosts. We investigated these characteristics using a specific cluster-DNA conjugate with the goal of developing a sensitive and selective biosensor. The silver cluster has a single violet absorption band (λ(max) = 400 nm), and its single-stranded DNA host has two domains that stabilize this cluster and hybridize with target oligonucleotides. These target analytes transform the weakly emissive violet cluster to a new chromophore with blue-green absorption (λ(max) = 490 nm) and strong green emission (λ(max) = 550 nm). Our studies consider the synthesis, cluster size, and DNA structure of the precursor violet cluster-DNA complex. This species preferentially forms with relatively low amounts of Ag(+), high concentrations of the oxidizing agent O2, and DNA strands with ≳20 nucleotides. The resulting aqueous and gaseous forms of this chromophore have 10 silvers that coalesce into a single cluster. This molecule is not only a chromophore but also an adduct that coordinates multiple nucleobases. Large-scale DNA conformational changes are manifested in a 20% smaller hydrodynamic radius and disrupted nucleobase stacking. Multidentate coordination also stabilizes the single-stranded DNA and thereby inhibits hybridization with target complements. These observations suggest that the silver cluster-DNA conjugate acts like a molecular beacon but is distinguished because the cluster chromophore not only sensitively signals target analytes but also stringently discriminates against analogous competing analytes.

  15. Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

    PubMed Central

    Rahman, Md. Mahbubur; Li, Xiao-Bo; Lopa, Nasrin Siraj; Ahn, Sang Jung; Lee, Jae-Joon

    2015-01-01

    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. PMID:25664436

  16. Identification of Lotus rhizobia by direct DNA hybridization of crushed root nodules

    SciTech Connect

    Cooper, J.E.; Bjourson, A.J.; Thompson, J.K.

    1987-07-01

    Hybridization of crushed Lotus pedunculatus root nodules with /sup 32/P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequency with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.

  17. Gelatin methacrylate (GelMA) mediated electrochemical DNA biosensor for DNA hybridization.

    PubMed

    Topkaya, Seda Nur

    2015-02-15

    In this study, an electrochemical biosensor system for the detection of DNA hybridization by using gelatin methacrylate (GelMA) modified electrodes was developed. Electrochemical behavior of GelMA modified Pencil Graphite Electrode (PGE) that serve as a functional platform was investigated by using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) and compared with those of the bare PGE. Hybridization was achieved in solution phase and guanine oxidation signal changes were evaluated. The decrease in the guanine oxidation peak currents at around +1.0 V was used as an indicator for the DNA hybridization. Also, more interestingly GelMA intrinsic oxidation peaks at around +0.7 V changed substantially by immobilization of different oligonucleotides such as probe, hybrid and control sequences to the electrode surface. It is the first study of using GelMA as a part of an electrochemical biosensor system. The results are very promising in terms of using GelMA as a new DNA hybridization indicator. Additionally, GelMA modified electrodes could be useful for detecting ultra low quantity of oligonucleotides by providing mechanical support to the bio-recognition layer. The detection limit of this method is at present 10(-12)mol. Signal suppressions were increased from 50% to 93% for hybrid with using GelMA when it was compared to bare electrode which facilitates the hybridization detection.

  18. How does RNase H recognize a DNA.RNA hybrid?

    PubMed

    Nakamura, H; Oda, Y; Iwai, S; Inoue, H; Ohtsuka, E; Kanaya, S; Kimura, S; Katsuda, C; Katayanagi, K; Morikawa, K

    1991-12-15

    The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

  19. Degradation of DNA RNA Hybrids by Ribonuclease H and DNA Polymerases of Cellular and Viral Origin

    PubMed Central

    Keller, Walter; Crouch, Robert

    1972-01-01

    Ribonuclease H from human KB cells, chick embryos, calf thymus, avian myeloblastosis virus, and Rous associated virus specifically degrades the RNA of DNA·RNA hybrids, producing mono- and oligoribonucleotides terminated in 5′-phosphates. The cellular RNase H is an endonuclease, whereas the viral enzyme appears to be an exonuclease. Viral DNA polymerase and RNase H copurify through all separation steps. Therefore, RNase H activity is an intrinsic part of the viral DNA polymerase. DNA·RNA hybrids are also degraded by nucleases associated with cellular DNA polymerases and by exonuclease III. However, these nucleases differ from RNase H in their ability to degrade both strands of DNA·RNA hybrids. Images PMID:4343966

  20. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  1. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

    1995-11-28

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

  2. Proximity hybridization regulated DNA biogate for sensitive electrochemical immunoassay.

    PubMed

    Ren, Kewei; Wu, Jie; Zhang, Yue; Yan, Feng; Ju, Huangxian

    2014-08-01

    An electrochemical DNA biogate was designed for highly sensitive homogeneous electrochemical immunoassay by combining target-induced proximity hybridization with a mesoporous silica nanoprobe (MSN). The electroactive methylene blue (MB) was sealed in the inner pores of MSN with single-stranded DNA. In the presence of target protein and two DNA-labeled antibodies, the formed proximate complex could hybridize with the DNA strand to form a rigid double-stranded structure and thus open the biogate, which led to the release of MB entrapped in the MSN. The target protein-dependent amount of released MB could be conveniently monitored with a screen-printed carbon electrode. Moreover, the detachment process of MB could be further amplified with an in situ enzymatic recycling binding of the proximate complex with the single-stranded DNA. Using prostate-specific antigen as a model target, the proposed assay showed a wide detection range from 0.002 to 100 ng mL(-1) with a detection limit of 1.3 pg mL(-1). This strategy was simple and universal for various analytes with different affinity ligands. This method possessed great potential for convenient point-of-care testing and commercial application.

  3. Impedimetric detection for DNA hybridization within microfluidic biochips.

    PubMed

    Lingerfelt, Louise; Karlinsey, James; Landers, James; Guiseppi-Elie, Anthony

    2007-01-01

    A fully integrated biochip for the performance of microfluidic-based DNA bioassays is presented. A microlithographically fabricated circumferential interdigitated electrode array of 1- to 5-microm critical line and space dimensions, with associated large area counterelectrode (1000 x WE) and reference electrode (Ag/AgCl), has been developed as a four-electrode system for the electrochemical detection of DNA hybridization using any of the techniques of amperometry, voltammetry, potentiometry, and impedimetry. This is presented as an alternative to optical detection with an emphasis on label-free impedimetric detection of hybridization. A micro total analysis system (microTAS) is presented, using fluidic channels to connect integrated reaction domains with downstream electrochemical detection. This is accomplished by bonding a patterned poly(dimethylsiloxane) (PDMS) substrate to the biochip or by adhesive bonding of the chip to channels fabricated within glass and plastic microfluidic cards, adding increased functionality to the device.

  4. A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

    SciTech Connect

    Kovtun, Oleg; Ross, Emily; Tomlinson, Ian; Rosenthal, Sandra

    2012-01-01

    Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs).We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

  5. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    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.

  6. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    PubMed

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors.

  7. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    PubMed

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. PMID:27612755

  8. Dual enzyme electrochemical coding for detecting DNA hybridization.

    PubMed

    Wang, Joseph; Kawde, Abdel-Nasser; Musameh, Mustafa; Rivas, Gustavo

    2002-10-01

    Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.

  9. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

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

    PubMed Central

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe; Nielsen, Katrine E.; Højland, Torben; Wengel, Jesper; Petersen, Michael

    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 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 sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both 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 a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface. PMID:21062815

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

    PubMed

    Toshima, Kazunobu; Okuno, Yukiko; Matsumura, Shuichi

    2003-10-01

    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.

  12. Label-free photoelectrochemical strategy for hairpin DNA hybridization detection on titanium dioxide electrode

    SciTech Connect

    Lu Wu; Wang Geng; Jin Yan; Yao Xin; Hu Jianqiang; Li Jinghong

    2006-12-25

    A new photoelectrochemical strategy for hairpin DNA hybridization was devised, in which TiO{sub 2} served as the anchor and signal transducer, and no label or redox couples were required. Once the hybridization between hairpin DNA probe and target DNA occurred, the photocurrent would decrease, utilizing which the sequence of the target DNA could be identified. The sequence specificity experiment showed that one or more mismatches of DNA bases could be discriminated. This photoelectrochemical method would be a potential tool in DNA hybridization detection due to its great advantages: label-free, high sensitivity, specific recognition, low cost, and easy fabrication.

  13. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    PubMed Central

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

    PubMed Central

    Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

    2014-01-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  16. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

    PubMed

    Chan, Yujia A; Aristizabal, Maria J; Lu, Phoebe Y T; Luo, Zongli; Hamza, Akil; Kobor, Michael S; Stirling, Peter C; Hieter, Philip

    2014-04-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  17. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA

    PubMed Central

    Case, Mary E.; Schweizer, Michael; Kushner, Sidney R.; Giles, Norman H.

    1979-01-01

    An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2+ gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2- mutation and an arom-9- mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2+ and had normal levels of catabolic dehydroquinase. DNA·DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2+ gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of the qa-2- gene by the qa-2+ gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2+ gene accompanied by inactivation of the adjacent qa-4+ gene, and (iii) insertion of a normal qa-2+ gene at an unlinked site in the N. crassa genome. This newly integrated qa-2+ genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2+ transformants are unlinked to the qa-2- gene of the recipient. Images PMID:159454

  18. Detection of DNA targets hybridized to solid surfaces using optical images of liquid crystals.

    PubMed

    Lai, Siok Lian; Tan, Wei Ling; Yang, Kun-Lin

    2011-09-01

    In this paper, we report a method of detecting DNA targets hybridized to a solid surface by using liquid crystals (LC). The detection principle is based on different interference colors of LC supported on surfaces decorated with single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). However, the contrast between the ssDNA and dsDNA is not obvious, unless DNA-streptavidin complexes are introduced to the dsDNA to increase the surface mass density. Two different approaches of introducing streptavidin to the system are studied and compared. We find that by premixing the biotin-labeled DNA targets with streptavidin prior to the DNA hybridization, branched-streptavidin complexes are formed and clear LC signal can be observed. This LC-based DNA detection principle represents an important step toward the development of a simple, instrument- and fluorophore-free DNA detection method.

  19. Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization.

    PubMed

    Jones, F S; Grimberg, J I; Fischer, S G; Ford, J P

    1985-01-01

    We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Crowding-Induced Hybridization of Single DNA Hairpins.

    PubMed

    Baltierra-Jasso, Laura E; Morten, Michael J; Laflör, Linda; Quinn, Steven D; Magennis, Steven W

    2015-12-30

    It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems. PMID:26654490

  2. Discrimination of DNA hybridization using chemical force microscopy.

    PubMed Central

    Mazzola, L T; Frank, C W; Fodor, S P; Mosher, C; Lartius, R; Henderson, E

    1999-01-01

    Atomic force microscopy (AFM) can be used to probe the mechanics of molecular recognition between surfaces. In the application known as "chemical force" microscopy (CFM), a chemically modified AFM tip probes a surface through chemical recognition. When modified with a biological ligand or receptor, the AFM tip can discriminate between its biological binding partner and other molecules on a heterogeneous substrate. The strength of the interaction between the modified tip and the substrate is governed by the molecular affinity. We have used CFM to probe the interactions between short segments of single-strand DNA (oligonucleotides). First, a latex microparticle was modified with the sequence 3'-CAGTTCTACGATGGCAAGTC and epoxied to a standard AFM cantilever. This DNA-modified probe was then used to scan substrates containing the complementary sequence 5'-GTCAAGATGCTACCGTTCAG. These substrates consisted of micron-scale, patterned arrays of one or more distinct oligonucleotides. A strong friction interaction was measured between the modified tip and both elements of surface-bound DNA. Complementary oligonucleotides exhibited a stronger friction than the noncomplementary sequences within the patterned array. The friction force correlated with the measured strength of adhesion (rupture force) for the tip- and array-bound oligonucleotides. This result is consistent with the formation of a greater number of hydrogen bonds for the complementary sequence, suggesting that the friction arises from a sequence-specific interaction (hybridization) of the tip and surface DNA. PMID:10354420

  3. Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

    PubMed

    Yao, Gang; Fang, Xiaohong; Yokota, Hiroaki; Yanagida, Toshio; Tan, Weihong

    2003-11-21

    We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

  4. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

    PubMed Central

    Hamperl, Stephan; Cimprich, Karlene A.

    2014-01-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

  5. Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

    PubMed Central

    Bjourson, A. J.; Cooper, J. E.

    1988-01-01

    Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes. Images PMID:16347782

  6. Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization.

    PubMed

    Hannon, K; Johnstone, E; Craft, L S; Little, S P; Smith, C K; Heiman, M L; Santerre, R F

    1993-08-01

    We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.

  7. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    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. PMID:26594643

  8. Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

    PubMed

    Brutlag, D; Fry, K; Nelson, T; Hung, P

    1977-03-01

    Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes. PMID:403010

  9. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed Central

    Wirth, D F; Pratt, D M

    1982-01-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. Images PMID:6960359

  10. Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions.

    PubMed

    Wirth, D F; Pratt, D M

    1982-11-01

    Kinetoplast DNA (kDNA) was isolated from various species of the protozoic parasite Leishmania and analyzed by nucleic acid hybridization to detect species-related heterogeneity of kDNA. Purified DNA isolated from L. mexicana and L. braziliensis displayed no homology in nucleic acid hybridization studies. These results confirmed that rapid kDNA sequence change and evolution is occurring in New World species of Leishmania and suggested that such isolated kDNA could be used as a specific hybridization probe for the rapid identification of Leishmania species by using whole organisms. This work further demonstrates that such species-specific identification is feasible on isolated Leishmania promastigotes and, more important, directly on tissue touch blots derived from the cutaneous lesion. Thus, specific hybridization of isolated kDNA provides the basis for a rapid, accurate method for the diagnosis of human leishmaniasis directly from infected tissue. PMID:6960359

  11. Microbiota of deciduous endodontic infections analyzed by MDA and Checkerboard DNA-DNA hybridization

    PubMed Central

    Tavares, WLF; de Brito, LC Neves; Teles, RP; Massara, MLA; Sobrinho, AP Ribeiro; Haffajee, AD; Socransky, SS; Teles, FR

    2011-01-01

    Aims To evaluate the microbiota of endodontic infections in deciduous teeth by checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). Methodology Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interadicular bone resorption were collected and 32 were analyzed, with 3 individuals contributing 2 samples; these were MDA- amplified and analyzed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species; and the mean proportion of each bacterial taxon present across all samples were computed. Results The mean amount of DNA in the samples prior to amplification was 5.2 (± 4.7) ng and 6.1 (± 2.3) μg after MDA. The mean number of species detected per sample was 19 (± 4) (range: 3–66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. Conclusion Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens among the most prominent species detected. PMID:21083570

  12. Immunofluorescent characterization of DNA . RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, sciaridae).

    PubMed

    Büsen, W; Amabis, J M; Leoncini, O; Stollar, B D; Lara, F J

    1982-01-01

    We have studied the distribution of DNA X RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA X RNA hybrids using the immunofluorescence technique. Fixed polytene chromosomes of the sciarid Trichosia pubescens (Diptera) show distinct, stage-specific labelling patterns throughout larval development. Controls for the staining procedure - including preincubation with hybrid-specific endoribonuclease H - prove that DNA X RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilize all chromosomal proteins, indicating that some proteins have to be removed to make the antigens accessible to antibody. Certain fixation conditions may also cause local denaturation of chromosomal DNA, and some hybrids may possibly form during specimen preparation. After incorporation of radioactive uridine, a combination of phase contrast, fluorescent, and autoradiographic images of one and the same chromosomal preparation demonstrates that hybrid fluorescence is confined to transcriptionally active regions. Two puff classes can be distinguished. The first binds antibody and includes most RNA puffs and all DNA puffs so far studied; the second, comprising some RNA puffs, does not show bright fluorescence in spite of the fact that RNA synthesis is high as revealed by 3H-uridine incorporation. DNA X RNA hybrids are not found at DNA puff sites during the DNA amplification period; these sites contain detectable hybrids only when transcription is taking place. - Combination of the fluorescent technique with its excellent resolution and autoradiography should be helpful in studying detailed topological aspects of transcriptionally active chromosomal regions.

  13. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    NASA Technical Reports Server (NTRS)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  14. Electrical detection of DNA immobilization and hybridization by streaming current measurements in microchannels

    NASA Astrophysics Data System (ADS)

    Martins, D. C.; Chu, V.; Prazeres, D. M. F.; Conde, J. P.

    2011-10-01

    Label-free electrical detection of surface DNA immobilization and hybridization via streaming current measurements in a microchannel is demonstrated. Streaming currents generated by the flow of deionised water through a polydimethysiloxane microchannel sealed on glass are measured using integrated Au electrodes and are sensitive to the density and polarity of the charge on the channel surface. An in-channel DNA hybridization protocol was developed. Streaming currents were monitored after each of protocol steps. The technique was applied to label free recognition of DNA hybridization and could distinguish between assays with complementary and non-complementary DNA strands.

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

    PubMed Central

    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 bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed. Images PMID:6406432

  16. Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates.

    PubMed

    Liu, Shu-Feng; Li, Yong-Fang; Li, Jin-Ru; Jiang, Long

    2005-11-15

    Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.

  17. Rapid DNA hybridization analysis using a PDMS microfluidic sensor and a molecular beacon.

    PubMed

    Kim, Sungyong; Chen, Lingxin; Lee, Sangyeop; Seong, Gi Hun; Choo, Jaebum; Lee, Eun Kyu; Oh, Chil-Hwan; Lee, Sanghoon

    2007-04-01

    A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.

  18. Simulation-Guided DNA Probe Design for Consistently Ultraspecific Hybridization

    PubMed Central

    Wang, J. Sherry; Zhang, David Yu

    2015-01-01

    Hybridization of complementary sequences is one of the central tenets of nucleic acid chemistry; however, the unintended binding of closely related sequences limits the accuracy of hybridization-based approaches for analyzing nucleic acids. Thermodynamics-guided probe design and empirical optimization of reaction conditions have been used to enable discrimination of single nucleotide variants, but typically these approaches provide only an approximate 25-fold difference in binding affinity. Here we show that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters. Simulation-guided probe systems designed against 44 different target single nucleotide variants sequences showed between 200- and 3000-fold (median 890) higher binding affinity than their corresponding wildtype sequences. As a demonstration of the usefulness of this simulation-guided design approach we developed probes which, in combination with PCR amplification, we use to detect low concentrations of variant alleles (1%) in human genomic DNA. PMID:26100802

  19. Numerical modeling of DNA-chip hybridization with chaotic advection

    PubMed Central

    Raynal, Florence; Beuf, Aurélien; Carrière, Philippe

    2013-01-01

    We present numerical simulations of DNA-chip hybridization, both in the “static” and “dynamical” cases. In the static case, transport of free targets is limited by molecular diffusion; in the dynamical case, an efficient mixing is achieved by chaotic advection, with a periodic protocol using pumps in a rectangular chamber. This protocol has been shown to achieve rapid and homogeneous mixing. We suppose in our model that all free targets are identical; the chip has different spots on which the probes are fixed, also all identical, and complementary to the targets. The reaction model is an infinite sink potential of width dh, i.e., a target is captured as soon as it comes close enough to a probe, at a distance lower than dh. Our results prove that mixing with chaotic advection enables much more rapid hybridization than the static case. We show and explain why the potential width dh does not play an important role in the final results, and we discuss the role of molecular diffusion. We also recover realistic reaction rates in the static case. PMID:24404027

  20. Simulation-guided DNA probe design for consistently ultraspecific hybridization

    NASA Astrophysics Data System (ADS)

    Wang, Juexiao Sherry; Zhang, David Yu

    2015-07-01

    Hybridization of complementary sequences is one of the central tenets of nucleic acid chemistry; however, the unintended binding of closely related sequences limits the accuracy of hybridization-based approaches to analysing nucleic acids. Thermodynamics-guided probe design and empirical optimization of the reaction conditions have been used to enable the discrimination of single-nucleotide variants, but typically these approaches provide only an approximately 25-fold difference in binding affinity. Here we show that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters. Simulation-guided probe systems designed against 44 sequences of different target single-nucleotide variants showed between a 200- and 3,000-fold (median 890) higher binding affinity than their corresponding wild-type sequences. As a demonstration of the usefulness of this simulation-guided design approach, we developed probes that, in combination with PCR amplification, detect low concentrations of variant alleles (1%) in human genomic DNA.

  1. Hybrid magnetic nanoparticle/nanogold clusters and their distance-dependent metal-enhanced fluorescence effect via DNA hybridization

    NASA Astrophysics Data System (ADS)

    GuThese Authors Contributed Equally To This Study., Xuefan; Wu, Youshen; Zhang, Lingze; Liu, Yongchun; Li, Yan; Yan, Yongli; Wu, Daocheng

    2014-07-01

    To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV)-visible (vis) absorption spectra of the samples were recorded with a UV-2600 spectrophotometer. Fluorescence spectra and the MEF effect were recorded using a spectrophotofluorometer, and lifetimes were determined using a time-correlated single photon counting apparatus. The prepared HMNCs were stable in aqueous solutions and had an average diameter of 87 +/- 3.2 nm, with six to eight AuNPs around a single Fe3O4 nanoparticle. Fluorescein isothiocyanate (FITC) tagged DNA-HMNC conjugates exhibited a significant MEF effect and could accurately detect specific DNA sequences after DNA hybridization. This result indicates their various potential applications in sensors and biomedical fields.To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV

  2. Single-Stranded DNA Catalyzes Hybridization of PCR-Products to Microarray Capture Probes

    PubMed Central

    Dally, Simon; Rupp, Steffen; Lemuth, Karin; Hartmann, Stefan C.; Hiller, Ekkehard; Bailer, Susanne M.; Knabbe, Cornelius; Weile, Jan

    2014-01-01

    Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays. PMID:25025686

  3. DNA damage, repair and photoadaptation in a Xiphophorus fish hybrid.

    PubMed

    Mitchell, David L; Paniker, Lakshmi; Douki, Thierry

    2009-01-01

    Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F(1) hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.

  4. In vitro flow cytometry-based screening platform for cellulase engineering

    PubMed Central

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  5. In vitro flow cytometry-based screening platform for cellulase engineering.

    PubMed

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  6. The microwave sensing of DNA hybridization using carbon nanotubes decorated with gold nanoislands

    NASA Astrophysics Data System (ADS)

    Cismaru, Alina; Dragoman, Mircea; Radoi, Antonio; Dinescu, A.; Dragoman, Daniela

    2012-04-01

    The hybridization of the deoxyribonucleic acid (DNA) is detected with the help of electromagnetic band gap resonator. The resonance frequency of the unloaded resonator f0=16.07 GHz is shifted to the left at 11.49 GHz when the resonator is loaded with single-stranded DNA anchored to gold nanoislands decorating bamboo-shaped carbon nanotubes deposited on the resonator. Further, single stranded DNA is hybridized and the resonator frequency is shifted to 14.16 GHz for double-stranded DNA. So, the frequency span of the two DNA states are separated by a span of 2.6 GHz in the band 11.5-16.07 GHz due to the very different electrical permittivity values of single- and double-stranded DNA. Thus, the hybridization of DNA is detected unambiguously.

  7. Controlling microarray DNA hybridization efficiency by probe-surface distance and external surface electrostatics

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

    DNA microarrays are analytical devices designed to determine the composition of multicomponent solutions of nucleic acids, DNA or RNA. These devices are promising technology for diverse applications, including sensing, diagnostics, and drug/gene delivery. Here, we modify a hybridization adsorption isotherm to study the effects of probe-surface distance and the external electrostatic fields, on the oligonucleotide hybridization in microarray and how these effects are varies depending on surface probe density and target concentration. This study helps in our understanding on-surface hybridization mechanisms, and from it we can observe a significant effect of the probe-surface distance, and the external electrostatic fields, on the hybridization yield. In addition we present a simple new criteria to control the oligonucleotide hybridization efficiency by providing a chart illustrating the effects of all factors on the DNA-hybridization efficiency.

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

    PubMed

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

    2016-09-01

    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. PMID:27526861

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

    SciTech Connect

    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.

  10. Hybrid magnetic nanoparticle/nanogold clusters and their distance-dependent metal-enhanced fluorescence effect via DNA hybridization.

    PubMed

    Gu, Xuefan; Wu, Youshen; Zhang, Lingze; Liu, Yongchun; Li, Yan; Yan, Yongli; Wu, Daocheng

    2014-08-01

    To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV)-visible (vis) absorption spectra of the samples were recorded with a UV-2600 spectrophotometer. Fluorescence spectra and the MEF effect were recorded using a spectrophotofluorometer, and lifetimes were determined using a time-correlated single photon counting apparatus. The prepared HMNCs were stable in aqueous solutions and had an average diameter of 87 ± 3.2 nm, with six to eight AuNPs around a single Fe3O4 nanoparticle. Fluorescein isothiocyanate (FITC) tagged DNA-HMNC conjugates exhibited a significant MEF effect and could accurately detect specific DNA sequences after DNA hybridization. This result indicates their various potential applications in sensors and biomedical fields.

  11. DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

    PubMed Central

    Toukdarian, A E; Lidstrom, M E

    1984-01-01

    DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight. Images PMID:6321444

  12. Structure and DNA Hybridization Properties of Mixed Nucleic Acid/Maleimide-Ethylene Glycol Monolayers

    SciTech Connect

    Lee,C.; Nguyen, P.; Grainger, D.; Gamble, L.; Castner, D.

    2007-01-01

    The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s {yields} {pi}* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the 'high-density' probe surface than on the 'high-efficiency' probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.

  13. Mammalian mitochondrial DNA replication intermediates are essentially duplex, but contain extensive tracts of RNA/DNA hybrid

    PubMed Central

    Pohjoismäki, Jaakko L. O.; Holmes, J. Bradley; Wood, Stuart R.; Yang, Ming-Yao; Yasukawa, Takehiro; Reyes, Aurelio; Laura, J. Bailey; Cluett, Tricia J.; Goffart, Steffi; Willcox, Smaranda; Rigby, Rachel E.; Jackson, Andrew P.; Spelbrink, Johannes N.; Griffith, Jack D.; Crouch, Robert J.; Jacobs, Howard T.

    2010-01-01

    We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mtDNA replication intermediates (mtRIs) are essentially duplex throughout their length, but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mtDNA replication involving RNA incorporation throughout the lagging strand (RITOLS). PMID:20184890

  14. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis.

    PubMed

    Mankan, Arun K; Schmidt, Tobias; Chauhan, Dhruv; Goldeck, Marion; Höning, Klara; Gaidt, Moritz; Kubarenko, Andrew V; Andreeva, Liudmila; Hopfner, Karl-Peter; Hornung, Veit

    2014-12-17

    Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2'-5'), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.

  15. Biological investigation using a shear horizontal surface acoustic wave sensor: small "click generated" DNA hybridization detection.

    PubMed

    Zerrouki, Chouki; Fourati, Najla; Lucas, Romain; Vergnaud, Julien; Fougnion, Jean-Marie; Zerrouki, Rachida; Pernelle, Christine

    2010-12-15

    We have used a 104 MHz lithium tantalate (LiTaO(3)) surface acoustic wave (SAW) sensor to investigate DNA probes grafting and their further hybridization with natural and click generated (Cg-DNA) oligonucleotides. Natural DNA targets of different strand lengths, tosyl-di(tri, tetra) thymidine and azido-di(tri, tetra) thymidine oligonucleotides were tested. In our case, and besides the follow-up of a 34mer DNA hybridization, we detected complementarity between natural DNA probes and azido-tetra-thymidine for the first time, whereas previous hybridization studies reported a minimal of 10-mer oligonucleotides recognition length. We also demonstrated that contrarily to natural DNA, the synthesized oligonucleotides present stable bonds with complementary DNA strands. Frequency responses of both grafting and hybridization have shown the same shape: an exponential decay with different time constants, (187±1)s and (68±19) s for grafting and hybridization respectively. We have also shown that recognition between DNA strands and tetranucleotide analogues is comparable to natural 34mer DNA bases and presents the same time constant within uncertainties.

  16. Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

    PubMed Central

    Mankan, Arun K; Schmidt, Tobias; Chauhan, Dhruv; Goldeck, Marion; Höning, Klara; Gaidt, Moritz; Kubarenko, Andrew V; Andreeva, Liudmila; Hopfner, Karl-Peter; Hornung, Veit

    2014-01-01

    Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA. PMID:25425575

  17. Assembly of DNA-functionalized nanoparticles in alcoholic solvents reveals opposite thermodynamic and kinetic trends for DNA hybridization.

    PubMed

    Smith, Brendan D; Liu, Juewen

    2010-05-12

    DNA has been a key molecule in biotechnology and nanotechnology. To date, the majority of the experiments involving DNA have been performed in aqueous solutions, which may be related to the perception that DNA hybridization is slower and less stable in organic solvents. All studies on the effect of organic solvents have focused on thermodynamic properties such as DNA melting temperature and the B-to-A form transition for very long DNAs, but not on the hybridization kinetics of short synthetic DNAs. We employed DNA-functionalized gold nanoparticles (AuNPs) as a model system and found that if the alcohol content is less than approximately 30%, more alcohol leads to a faster DNA hybridization, although with a decreased melting temperature. The generality of this observation was independently verified with two molecular beacon systems (in the absence of AuNPs) using fluorophore and quencher-labeled DNAs. With 25% ethanol, the hybridization rates are three to four times faster than in the case with water. This discovery will extend the application of DNA bio- and nanotechnology to organic solvents with improved performance.

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

    PubMed

    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.

  19. [Evaluation of DNA-DNA hybridization method for identification of mycobacteria using a colorimetric microplate kit].

    PubMed

    Yamazaki, T; Takahashi, H; Nakamura, R M

    1993-01-01

    DNA-DNA hybridization was applied for identification of mycobacteria and developed as a kit "microplate hybridization kit" (refers to MPHD) by Kobayashi Pharmaceutical Co. We received test samples of the microplates from the company and examined them for their and reliability using 180 mycobacterial strains of 21 species kept in our laboratory. The results of identification by MPHD were 100% identical to those of biochemical identification in the type or reference strains of mycobacteria, showing good reliability of MPHD method. Among clinical isolates, there were six M. tuberculosis strains which did not show typical characteristics for M. tuberculosis, i.e., niacin test negative or nitrate reduction weak positive, but all of these were identified as M. tuberculosis complex by MPHD method. Some strains from clinical isolates showed difference in identification between MPHD and biochemical methods: M. avium complex, identified biochemically were divided into M. avium and M. intracellulare by MPHD, M. fortuitum complex by biochemical identification were distinguished as M. fortuitum and M. chelonae by MPHD. Further, M. chelonae were separated into M. chelonae subsp. chelonae and M. chelonae subsp. abscessus by MPHD. M. peregrinum has been considered as a synonym of M. fortuitum, but we could distinguish M. peregrinum from M. fortuitum clearly by MPHD method. Thus, it is suggested that M. peregrinum and M. fortuitum are different species. Keys for getting reliable results using the MPHD kit are: (1) appropriate amount of bacteria for use, (2) purification of DNA, (3) enough deproteinization, and (4) appropriate timing to read colorimetry measurement of the plate. PMID:8437424

  20. Measuring antibody neutralization of dengue virus (DENV) using a flow cytometry-based technique.

    PubMed

    de Alwis, Ruklanthi; de Silva, Aravinda M

    2014-01-01

    Dengue virus (DENV) is an emerging virus that threatens over two-third of the world's population. The specific diagnosis of dengue infection by serology is based on assays that detect DENV-specific antibodies including neutralizing antibodies (Abs). Neutralizing Abs are an important, if not the main, mechanism of protection from natural dengue virus (DENV) infection as well. The current gold-standard assay for measuring neutralizing Ab responses against DENV is the plaque reduction neutralization assay (PRNT). However, this assay is slow and laborious and utilizes physiologically irrelevant cell lines. Here, we describe a relatively high-throughput, flow cytometry-based neutralization assay for DENV that has been optimized for use with a human monocytic suspension cell line, U937 + DC-SIGN, or the more commonly used adherent monkey kidney cells, Vero-81. PMID:24696329

  1. Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

    PubMed Central

    Livshits, M A; Mirzabekov, A D

    1996-01-01

    A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization. Images FIGURE 1 PMID:8913616

  2. Influence of attachment strategy on the thermal stability of hybridized DNA on gold surfaces.

    PubMed

    Petty, Tyler J; Wagner, Caleb E; Opdahl, Aric

    2014-12-23

    The thermal stabilities of double-stranded DNA hybrids immobilized on gold surfaces are shown to be significantly affected by the conformation of the hybrid. To analyze this behavior, DNA probes were immobilized using attachment strategies where the nucleotides within the strand had varying levels of interactions with the gold substrate. The abilities of these probes to form double-stranded hybrids with solution DNA targets were evaluated by surface plasmon resonance (SPR) over a temperature range 25-60 °C. The measurements were used to construct thermal stability profiles for hybrids in each conformation. We observe that DNA hybrids formed with probe strands that interact extensively with the gold surface have stability profiles that are shifted lower by 5-10 °C compared to hybrids formed with end-tethered probes that have fewer interactions with the surface. The results provide an understanding of the experimental conditions in which these weaker DNA hybrids can form and show the additional complexity of evaluating denaturation profiles generated from DNA on surfaces.

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

    PubMed

    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.

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

    SciTech Connect

    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.

  5. Biomolecular hybrid of a conducting polymer with DNA: morphology, structure, and doping behavior.

    PubMed

    Dawn, Arnab; Nandi, Arun K

    2005-05-23

    A poly(o-methoxyaniline) (POMA)/DNA [weight fraction of DNA (W(DNA)) = 0.45] hybrid was prepared by mixing their solutions in sterilized double distilled water. The solution turned green upon aging for a longer time, and the doping of POMA by DNA was complete after about 15 d of aging. The doping was confirmed from the UV-vis spectra where the 599 nm peak of POMA(EB) disappeared and a new peak for a pi to localized polaron band-transition appeared. With increasing aging time the new peak gradually shifted from 674 nm at 3 h to 820 nm at 15 d of mixing and thereafter it remained constant. The absence of a free carrier tail in the UV-vis spectra indicated a coiled structure of POMA in the complex. Circular dichroism spectra of the hybrid solution indicated that the DNA conformation (double helical structure) remained unchanged in the hybrid. The SEM micrograph of the freeze-dried hybrid showed a needle-like morphology of the DNA dispersed in a polymer matrix and it was completely different from the fibrillar network morphology of pure DNA in the solid state. The TEM micrograph indicated a homogeneous dispersion of DNA fibrils in the POMA matrix. The melting temperature of the POMA-DNA hybrid showed an increase compared to that of pure DNA by 5 degrees C, probably caused by an electrostatic interaction between the DNA anion and the POMA radical cation generated in the doping process. WAXS investigations revealed that the DNA crystal structure remained unchanged in the hybrid whereas the POMA crystal structure might be lost. An FT-IR study suggested that interaction occurred between the phosphoric acid group of DNA and a nitrogen atom of POMA through proton transfer from the OH group of the former. A schematic model of the POMA-DNA complex randomly anchoring POMA chains with the DNA molecule was proposed. The dc conductivity of the POMA-DNA complex was found to be ca. 10(-7) S . cm(-1). Hence, this work describes a procedure for making a DNA-conducting polymer hybrid

  6. Complete mitochondrial DNA sequence analysis of Bison bison and bison-cattle hybrids: function and phylogeny.

    PubMed

    Douglas, Kory C; Halbert, Natalie D; Kolenda, Claire; Childers, Christopher; Hunter, David L; Derr, James N

    2011-01-01

    Complete mitochondrial DNA (mtDNA) genomes from 43 bison and bison-cattle hybrids were sequenced and compared with other bovids. Selected animals reflect the historical range and current taxonomic structure of bison. This study identified regions of potential nuclear-mitochondrial incompatibilities in hybrids, provided a complete mtDNA phylogenetic tree for this species, and uncovered evidence of bison population substructure. Seventeen bison haplotypes defined by 66 polymorphic sites were discovered, whereas 728 fixed differences and 86 non-synonymous mutations were identified between bison and bison-cattle hybrid sequences. The potential roles of the mtDNA genome in the function of hybrid animals and bison taxonomy are discussed.

  7. Maternal transmission of cytoplasmic DNA in interspecific hybrids of peat mosses, Sphagnum (Bryophyta).

    PubMed

    Natcheva, R; Cronberg, N

    2007-07-01

    The progeny of spontaneous interspecific hybrid sporophytes of Sphagnum were used to analyse the inheritance of cytoplasmic DNA. The analysis showed that only the female parent donated chloroplasts and mitochondria in Sphagnum hybrids. Thus, this is the first study demonstrating maternal cytoplasmic inheritance in a nonvascular land plant. This finding has important implications for phylogenetic reconstructions utilizing chloroplast and mitochondrial DNA sequences as well as for the evolution of cytoplasmic inheritance in relation to the life cycle of land plants.

  8. Stress response in Drosophila subobscura: DNA-RNA hybrids and transcriptional activity.

    PubMed

    Arbona, M; Cuenca, J B; de Frutos, R

    1992-01-01

    Immunofluorescent techniques have been used in the analysis of DNA-RNA hybrids occurrence and its relationship to transcriptional events on polytene chromosomes of Drosophila subobscura. We have studied the distribution of these hybrids on uninduced/induced chromosomes. Two different indirect immunofluorescence methods for the detection of DNA-RNA hybrids were used. Our data confirm the positive correlation between localization of DNA-RNA hybrids and transcriptional activity by following the Büsen et al procedure (1982). Using the other protocol, which allows chromosomal DNA-RNA to denature and renature, makes DNA-RNA hybrids detectable not exclusively in active chromosomal regions. Taking Büsen as method of choice, this technique allowed to localize the exact transcriptional active sites on puffs: hybrid fluorescence was restricted to marginal or central puff areas. Moreover, no correlation between fluorescence and puffs size was found. However, our studies on induced chromosomes indicate that: 1) the 15DE puff, previously described as t-puff, was not really a heat shock puff, since no transcriptional activity was detected; 2) hybrid fluorescence at 2C and 31CD regions was observed. No labelling was found in these loci in the autoradiography data, reported by other authors.

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

    SciTech Connect

    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.

  10. Biofunctionalized AlGaN/GaN high electron mobility transistor for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Thapa, Resham; Alur, Siddharth; Kim, Kyusang; Tong, Fei; Sharma, Yogesh; Kim, Moonil; Ahyi, Claude; Dai, Jing; Wook Hong, Jong; Bozack, Michael; Williams, John; Son, Ahjeong; Dabiran, Amir; Park, Minseo

    2012-06-01

    Label-free electrical detection of deoxyribonucleic acid (DNA) hybridization was demonstrated using an AlGaN/GaN high electron mobility transistor (HEMT) based transducer with a biofunctionalized gate. The HEMT DNA sensor employed the immobilization of amine-modified single strand DNA on the self-assembled monolayers of 11-mercaptoundecanoic acid. The sensor exhibited a substantial current drop upon introduction of complimentary DNA to the gate well, which is a clear indication of the hybridization. The application of 3 base-pair mismatched target DNA showed little change in output current characteristics of the transistor. Therefore, it can be concluded that our DNA sensor is highly specific to DNA sequences.

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

    PubMed

    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. <5 °C) for 2-3 days. Aiming to improve the reaction's turnover rate, we evaluated microwave irradiation with simultaneous cooling as potential energy source since this method has been widely used to accelerate various chemical and enzymatic reactions. However, our data indicated that microwave irradiation induced an inactivation of 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.

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

    NASA Astrophysics Data System (ADS)

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

    2006-01-01

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

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

    PubMed

    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.

  14. DNA-inorganic hybrid nanovaccine for cancer immunotherapy

    NASA Astrophysics Data System (ADS)

    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

  15. DNA-inorganic hybrid nanovaccine for cancer immunotherapy.

    PubMed

    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

    2016-03-28

    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.

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

    PubMed

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

    2016-04-01

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

  17. Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer.

    PubMed

    Pumera, Martin; Castañeda, Maria Teresa; Pividori, Maria Isabel; Eritja, Ramon; Merkoçi, Arben; Alegret, Salvador

    2005-10-11

    A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.

  18. Highly sensitive detection of DNA hybridization on commercialized graphene-coated surface plasmon resonance interfaces.

    PubMed

    Zagorodko, Oleksandr; Spadavecchia, Jolanda; Serrano, Aritz Yanguas; Larroulet, Iban; Pesquera, Amaia; Zurutuza, Amaia; Boukherroub, Rabah; Szunerits, Sabine

    2014-11-18

    Strategies employed to interface biomolecules with nanomaterials have considerably advanced in recent years and found practical applications in many different research fields. The construction of nucleic acid modified interfaces together with the label-free detection of hybridization events has been one of the major research focuses in science and technology. In this paper, we demonstrate the high interest of graphene-on-metal surface plasmon resonance (SPR) interfaces for the detection of DNA hybridization events in the attomolar concentration range. The strategy consists on the noncovalent functionalization of graphene-coated SPR interfaces with gold nanostars carrying single-stranded DNA (ssDNA). Upon hybridization with its complementary DNA, desorption of the nanostructures takes place and thus enables the sensitive detection of the DNA hybridization event. The DNA sensor exhibits a detection limit of ≈500 aM for complementary DNA with a linear dynamic range up to 10(-8) M. This label-free DNA detection platform should spur off new interest toward the use of commercially available graphene-coated SPR interfaces. PMID:25341125

  19. A Hybrid Computer Simulation to Generate the DNA Distribution of a Cell Population.

    ERIC Educational Resources Information Center

    Griebling, John L.; Adams, William S.

    1981-01-01

    Described is a method of simulating the formation of a DNA distribution, on which statistical results and experimentally measured parameters from DNA distribution and percent-labeled mitosis studies are combined. An EAI-680 and DECSystem-10 Hybrid Computer configuration are used. (Author/CS)

  20. DNA reorientation on Au nanoparticles: label-free detection of hybridization by surface enhanced Raman spectroscopy.

    PubMed

    Papadopoulou, Evanthia; Bell, Steven E J

    2011-10-21

    DNA sequences attached to Au nanoparticles via thiol linkers stand up from the surface, giving preferential enhancement of the adenine ring breathing SERS band. Non-specific binding via the nucleobases reorients the DNA, reducing this effect. This change in intensity on reorientation was utilised for label-free detection of hybridization of a molecular beacon.

  1. PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

    PubMed

    Knuchel, M C; Graf, B; Schlaepfer, E; Kuster, H; Fischer, M; Weber, R; Cone, R W

    2000-02-01

    We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)

  2. Electric-field assisted immobilization and hybridization of DNA oligomers on thin-film microchips.

    PubMed

    Fixe, F; Branz, H M; Louro, N; Chu, V; Prazeres, D M F; Conde, J P

    2005-10-01

    Single, square voltage pulses in the microsecond timescale result in selective 5'-end covalent bonding (immobilization) of thiolated single-stranded (ss) DNA probes to a modified silicon dioxide flat surface and in specific hybridization of ssDNA targets to the immobilized probe. Immobilization and hybridization rates using microsecond voltage pulses at or below 1 V are at least 10(8) times faster than in the passive control reactions performed without electric field (E), and can be achieved with at least three differently functionalized thin-film surfaces on plastic or glass substrates. The systematic study of the effect of DNA probe and target concentrations, of DNA probe and target length, and the application of asymmetric pulses on E-assisted DNA immobilization and hybridization showed that: (1) the rapidly rising edge of the pulse is most critical to the E-assisted processes, but the duration of the pulse is also important; (2) E-assisted immobilization and hybridization can be performed with micrometre-sized pixels, proving the potential for use on microelectronic length scales, and the applied voltage can be scaled down together with the electrode spacing to as low as 25 mV; and (3) longer DNA chains reduce the yield in the E-assisted immobilization and hybridization because the density of physisorbed single-stranded DNA is reduced. The results show that the E-induced reactions can be used as a general method in DNA microarrays to produce high-density DNA chips (E-immobilization) and speed the microarray-based analysis (E-hybridization). PMID:20817972

  3. RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

    PubMed Central

    Rigby, Rachel E; Webb, Lauren M; Mackenzie, Karen J; Li, Yue; Leitch, Andrea; Reijns, Martin A M; Lundie, Rachel J; Revuelta, Ailsa; Davidson, Donald J; Diebold, Sandra; Modis, Yorgo; MacDonald, Andrew S; Jackson, Andrew P

    2014-01-01

    The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease. PMID:24514026

  4. Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

    PubMed

    Bahga, Supreet S; Han, Crystal M; Santiago, Juan G

    2013-01-01

    We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

  5. Detection and analysis of leptospiral DNA in early Leptospirosis by polymerase chain reaction and DNA hybridization with Digoxingenin-AMPPD

    NASA Astrophysics Data System (ADS)

    Bao, Lang; Yu, Ye-Rong; Terpstra, W. J.

    1994-07-01

    Fourteen serum specimens from patients with early Leptospirosis proven by blood culture and serological test were detected by PCR with the oligonucleotide primers obtained from a genomic library of leptospira interrogans. The amplified DNA were hybridized with the homologous DNA probe labeling with Digoxingenin-AMPPD. All of the samples revealed the presence of leptospira and the strong signals were visualized with homologous DNA probes hybridization. Negative and positive controls appeared correctly. The DNA fragment generated from PCR amplification homologically hybridized with the DNA of 16 strains of leptospira. The single recognized band (about 400 bps) from 6 out of the 16 strains has come out which are representative of the principal strains in Sichuan, China. The results demonstrated that PCR is an advanced diagnostic technique for early Leptospirosis. The treatment of samples is easy and has little risk of DNA loss and contamination. This is a considerable advantage over other detective techniques and can be available especially in China and other developing countries.

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

    PubMed

    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.

  7. Intense photoluminescence from dried double-stranded DNA and single-walled carbon nanotube hybrid

    SciTech Connect

    Ito, M.; Kobayashi, T.; Ito, Y.; Hayashida, T.; Nii, D.; Umemura, K.; Homma, Y.

    2014-01-27

    Semiconducting single-walled carbon nanotubes (SWNTs) show near-infrared photoluminescence (PL) when they are individually isolated. This was an obstacle to use photonic properties of SWNTs on a solid surface. We show that SWNTs wrapped with DNA maintain intense PL under the dry conditions. SWNTs are well isolated individually by DNA even when the DNA-SWNT hybrids are agglomerated. This finding opens up application of SWNTs to photonic devices.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

  10. Fluorescent in situ hybridization of mitochondrial DNA and RNA.

    PubMed

    Alán, Lukáš; Zelenka, Jaroslav; Ježek, Jan; Dlasková, Andrea; Ježek, Petr

    2010-01-01

    To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization. DNase I spread the signal over mitochondrial tubules. Future applications are discussed.

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

    SciTech Connect

    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.

  12. Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets.

    PubMed

    Kuhn, Heiko; Demidov, Vadim V; Coull, James M; Fiandaca, Mark J; Gildea, Brian D; Frank-Kamenetskii, Maxim D

    2002-02-13

    Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

  13. Simulation study of noncovalent hybridization of carbon nanotubes by single-stranded DNA in water.

    PubMed

    Martin, Willis; Zhu, Wusheng; Krilov, Goran

    2008-12-18

    Recent discovery that single-stranded DNA (ssDNA) binds to carbon nanotubes with high affinity to form soluble hybrids has received great attention as a promising approach to solving the long-standing problem of nanotube solubilization and separation. The mechanism of this process, including the nature of the DNA-nanotube interactions and the molecular structure of the hybrids is still not well understood. Here, we use all-atom replica-exchange molecular dynamics simulations to study the association of several ssDNA decamers with single-walled carbon nanotubes of different chirality in an aqueous environment. The oligonucleotides are found to readily adsorb onto the nanotube surface, after which they undergo a slow structural rearrangement. Cluster analysis of bound DNA conformations as well as population distribution maps computed as a function of several local and global order parameters show that the hybrids exhibit a complex morphology with DNA strands assuming a number of distinct backbone geometries, which depend on both DNA sequence and nanotube diameter. In contrast, the nucleotide bases are found to align parallel to the nanotube surface with a high degree of orientational order. While the binding appears to be primarily driven by energetically favorable pi-stacking of DNA bases onto the nanotube surface, equilibrium distribution of hybrid conformations is modulated by a complex interplay of forces, including the DNA conformational strain and solvent interactions. As a result, the hybrid free-energy landscapes are found to be rugged, with multiple low-lying minima separated by high barriers, several of which are significantly populated at room temperature. Qualitative differences are observed in free energy profiles of purine- and pyrimidine-based oligonucleotide sequences and are attributed to the difference in self-stacking propensity of the bases.

  14. DNA hybridization efficiency on concave surface nano-structure in hemispherical Janus nanocups.

    PubMed

    Kim, Hyonchol; Terazono, Hideyuki; Takei, Hiroyuki; Yasuda, Kenji

    2014-02-11

    We examined the effect of a concave structure on DNA hybridization efficiency using an inner surface of hemispherical Janus nanocups in the range from 140 to 800 nm. Target DNA was specifically immobilized onto the inner cup surface, hybridized with complementary DNA-attached 20 nm Au probes, and the number of the hybridized probes was counted by scanning electron microscopy. The hybridization density of the attached Au probes on 800 nm nanocups was 255 μm(-2), which was 0.57 times that on a flat surface, 449 μm(-2), and increased to 394 μm(-2) on a 140 nm cup, 0.88 times of a flat surface, as the cup size decreased. The local density of attached Au probes within the central 25% at the bottom of the 800 nm nanocups was 444 μm(-2), which was closer to that on a flat surface, and the tendency was the same for all sizes of cups, indicating that the size dependency of DNA hybridization efficiency on the concave structures were mostly affected by the lower efficiency of side wall hybridization.

  15. Layered zirconium phosphonate with inorganic–organic hybrid structure: Preparation and its assembly with DNA

    SciTech Connect

    Liu, Li-Min; Lu, Guo-Yuan; Jiang, Li-Ping; Zhu, Jun-Jie

    2014-07-01

    An aminoethoxy-functionalized zirconium phosphonate (Zr(O{sub 3}POCH{sub 2}CH{sub 2}NH{sub 2}){sub 2}·3H{sub 2}O), abbreviated as ZrRP (R=OCH{sub 2}CH{sub 2}NH{sub 2}), with layered structure has been synthesized. This layered compound possesses the characteristic of inorganic–organic hybrid, due to the covalently linked aminoethoxy in the host layer. The anion exchanged property of this zirconium phosphonate is suitable for the direct intercalation of negatively charged DNA, which is different from these reported zirconium phosphates or zirconium phosphonates. As a precursor, this prepared zirconium phosphonate was utilized to fabricate a novel DNA/ZrRP binary hybrid via a delamination-reassembly procedure. The release behavior of DNA from the DNA/ZrRP composite was investigated at different medium pH, because the combination between zirconium phosphonate sheets and DNA was pH-dependent sensitively. Moreover, the helical conformation of DNA was almost retained after the intercalation and release process. These properties of the DNA/ZrRP composite suggested the potential application of layered zirconium phosphonate as a non-viral vector in gene delivery. - Graphical abstract: The intercalation of DNA into zirconium phosphonate and the release of DNA from the interlayer of zirconium phosphonate. - Highlights: ●A layered aminoethoxy-functionalized zirconium phosphonate has been synthesized. ●DNA was intercalated directly into the prepared zirconium phosphonate. ●A novel zirconium phosphonate/DNA binary hybrid was fabricated. ●DNA can be reversibly released from the interlayer of zirconium phosphonate. ●The intercalation/release processes do not induce the denaturalization of DNA.

  16. DNA hybridization and phosphinothricin acetyltransferase gene sequence detection based on zirconia/nanogold film modified electrode

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Yang, Tao; Jiang, Chen; Jiao, Kui

    2008-05-01

    This study reports a novel electrochemical DNA biosensor based on zirconia (ZrO 2) and gold nanoparticles (NG) film modified glassy carbon electrode (GCE). NG was electrodeposited onto the glassy carbon electrode at 1.5 V, and then zirconia thin film on the NG/GCE was fabricated by cyclic voltammetric method (CV) in an aqueous electrolyte of ZrOCl 2 and KCl at a scan rate of 20 mV/s. DNA probes were attached onto the ZrO 2/NG/GCE due to the strong binding of the phosphate group of DNA with the zirconia film and the excellent biocompatibility of nanogold with DNA. CV and electrochemical impedance spectroscopy (EIS) were used to characterize the modification of the electrode and the probe DNA immobilization. The electrochemical response of the DNA hybridization was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. After the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA), the cathodic peak current of MB decreased obviously. The difference of the cathodic peak currents of MB between before and after the hybridization of the probe DNA was used as the signal for the detection of the target DNA. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene in the transgenic plants was detected with a detection range from 1.0 × 10 -10 to 1.0 × 10 -6 mol/L, and a detection limit of 3.1 × 10 -11 mol/L.

  17. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

    PubMed Central

    Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin; Hartono, Stella R; Chédin, Frédéric

    2015-01-01

    Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE. DOI: http://dx.doi.org/10.7554/eLife.08007.001 PMID:26182405

  18. DNA fingerprinting of Kentucky bluegrass cultivars and hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As a high polyploidy, apomictic, self-incompatible, perennial grass, Kentucky bluegrass has such complex genetic architecture that conducting standard Mendelian genetic selection is currently impossible. One large hurdle is the inability to differentiate true hybrids from other apomictic progenies....

  19. Efficient Formation of Site-Specific Protein-DNA Hybrids Using Copper-Free Click Chemistry.

    PubMed

    Mukhortava, Ann; Schlierf, Michael

    2016-07-20

    Protein-DNA hybrids have become increasingly popular molecular building blocks in bionanotechnology and single-molecule studies to synergistically combine the programmability of DNA with the chemical diversity of proteins. The growing demand for protein-DNA hybrids requires powerful strategies for their conjugation. Here, we present an efficient two-step method for protein-DNA assembly based on copper-free click chemistry. The method allows site-specificity and high coupling efficiency, while maintaining the conservation of protein activity. We compare our method to a commonly used protocol of direct linkage of maleimide-modified oligos. We demonstrate the significantly higher yield with a protein-DNA conjugate, which is analyzed using single-molecule force spectroscopy. PMID:27322198

  20. Flow Cytometry-Based Methods to Characterize Immune Senescence in Nonhuman Primates.

    PubMed

    Meyer, Christine; Haberthur, Kristen; Asquith, Mark; Messaoudi, Ilhem

    2015-01-01

    Flow cytometry is an invaluable technique that can be used to phenotypically and functionally characterize immune cell populations ex vivo. This technology has greatly advanced our ability to gain critical insight into age-related changes in immune function, commonly known as immune senescence. Rodents have been traditionally used to investigate the molecular mechanisms of immune senescence because they offer the distinct advantages of an extensive set of reagents, the presence of genetically modified strains, and a short lifespan that allows for longevity studies of short duration. More recently, nonhuman primates (NHPs), and specifically rhesus macaques, have emerged as a leading translational model to study various aspects of human aging. In contrast to rodents, they share significant genetic homology as well as physiological and behavioral characteristics with humans. Furthermore, rhesus macaques are a long-lived outbred species, which makes them an ideal translational model. Therefore, NHPs offer a unique opportunity to carry out mechanistic studies under controlled laboratory conditions (e.g., photoperiod, temperature, diet, and medications) in a species that closely mimics human biology. Moreover similar techniques (e.g., activity recording and MRI) can be used to measure physiological parameters in NHPs, making direct comparisons between NHP and human data sets possible. In addition, the outbred genetics of NHPs enables rigorous validation of research findings that goes beyond proof of principle. Finally, self-selection bias that is often unavoidable in human clinical trials can be completely eliminated with NHP studies. Here we describe flow cytometry-based methods to phenotypically and functionally characterize innate immune cells as well as T and B lymphocyte subsets from isolated peripheral blood mononuclear cells (PBMC) in rhesus macaques. PMID:26420709

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

    PubMed

    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

    2016-05-01

    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. PMID:27058116

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

    PubMed

    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.

  3. DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L. ) larvae

    SciTech Connect

    Keating, S.T.; Burand, J.P.; Elkinton, J.S. )

    1989-11-01

    Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. The hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.

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

    PubMed

    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.

  5. Nanoporous niobium oxide for label-free detection of DNA hybridization events.

    PubMed

    Choi, Jinsub; Lim, Jae Hoon; Rho, Sangchul; Jahng, Deokjin; Lee, Jaeyoung; Kim, Kyung Ja

    2008-01-15

    We found that DNA probes can be immobilized on anodically prepared porous niobium oxide without a chemical modification of both the DNA probes and the substrate. By using the porous niobium oxide with a positive surface charge, DNA hybridization events are detected on the basis of the blue-shift of a maximum absorption peak in UV-vis-NIR spectroscopy. The blue-shift is ascribed to the change of surface charge upon single- or double-stranded DNA. The method does not require a label and shows high sensitivity with the detection limit of the concentration of 1nM.

  6. Hybridization of potato spindle tuber viroid to cellular DNA of normal plants.

    PubMed

    Hadidi, A; Jones, D M; Gillespie, D H; Wong-Staal, F; Diener, T O

    1976-07-01

    Molecular hybridization experiments of (125)I-labeled potato spindle tuber viroid (PSTV) with DNA from uninfected or PSTV-infected tomato plants showed that infrequent DNA sequences complementary to PSTV exist in both uninfected and infected cells. DNA titration experiments revealed that at least 60% of PSTV is represented by sequences in DNA of several normal solanaceous host species. Phylogenetically diverse plants contain sequences related to less of the PSTV. PSTV-infected tomato or Gynura aurantiaca plants did not possess new PSTV sequences at detectable levels. These results support the hypothesis that PSTV may have originated from genes in normal solanaceous plants.

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

    PubMed

    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.

  8. Using a microfluidic device for 1 μl DNA microarray hybridization in 500 s

    PubMed Central

    Wei, Cheng-Wey; Cheng, Ji-Yen; Huang, Chih-Ting; Yen, Meng-Hua; Young, Tai-Horng

    2005-01-01

    This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 μl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA–DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 μl target was used to hybridize with an array that can hold 5000 probes. PMID:15891111

  9. Trivalent lanthanide ions do not cleave RNA in DNA-RNA hybrids

    SciTech Connect

    Kolasa, K.A.; Morrow, J.R.; Sharma, A.P. )

    1993-09-15

    Lanthanide(III) complexes rapidly catalyze cleavage of single-stranded RNA. RNA cleavage by lanthanide complexes is, however, dependent on RNA structure. A DNA-RNA hybrid formed by annealing a complementary oligodeoxynucleotide to t-RNA[sup phe] is found to be inert to cleavage by a europium(III) hexadentate Schiff base complex and by Eu(CO[sub 2]CH[sub 3])[sub 3]. Because DNA-RNA hybrids are important structures in antisense oligonucleotide strategies, these results may influence the design of antisense oligonucleotides with attached metal complex cleaving agents.

  10. Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

    PubMed

    Tsourkas, Andrew; Behlke, Mark A; Bao, Gang

    2002-12-01

    Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.

  11. Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

    PubMed

    Tsourkas, Andrew; Behlke, Mark A; Bao, Gang

    2003-03-15

    Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.

  12. Electrochemical transduction of DNA hybridization at modified electrodes by using an electroactive pyridoacridone intercalator.

    PubMed

    Bouffier, Laurent; Wang, Bingquan Stuart; Roget, André; Livache, Thierry; Demeunynck, Martine; Mailley, Pascal

    2014-02-01

    A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.

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

    PubMed

    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.

  14. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  15. A DNA--silver nanocluster probe that fluoresces upon hybridization.

    PubMed

    Yeh, Hsin-Chih; Sharma, Jaswinder; Han, Jason J; Martinez, Jennifer S; Werner, James H

    2010-08-11

    DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that "lights up" upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

  16. Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures.

    PubMed

    Koussa, Mounir A; Sotomayor, Marcos; Wong, Wesley P

    2014-05-15

    Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. PMID:24568941

  17. Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures

    PubMed Central

    Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.

    2014-01-01

    Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. PMID:24568941

  18. Hybridization of genomic DNA to microarrays: a challenge for the analysis of environmental samples.

    PubMed

    Avarre, Jean-Christophe; de Lajudie, Philippe; Béna, Gilles

    2007-05-01

    The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.

  19. DNA sequence determination by hybridization: A strategy for efficient large-scale sequencing

    SciTech Connect

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

    1993-06-11

    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. 22 refs., 3 figs.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection.

    PubMed

    Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

    2014-10-21

    The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.

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

    PubMed Central

    Liu, Jinhuai; Liu, Jinyun; Yang, Liangbao; Chen, Xing; Zhang, Meiyun; Meng, Fanli; Luo, Tao; Li, Minqiang

    2009-01-01

    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. PMID:22399999

  3. Highly sensitive electrochemical biosensor based on nonlinear hybridization chain reaction for DNA detection.

    PubMed

    Jia, Liping; Shi, Shanshan; Ma, Rongna; Jia, Wenli; Wang, Huaisheng

    2016-06-15

    In the present work we demonstrated an ultrasensitive detection platform for specific DNA based on nonlinear hybridization chain reaction (HCR) by triggering chain-branching growth of DNA dendrimers. HCR was initiated by target DNA (tDNA) and finally formed dendritic structure by self-assembly. The electrochemical signal was drastically enhanced by capturing multiple catalytic peroxidase with high-ordered growth. Electrochemical signals were obtained by measuring the reduction current of oxidized 3, 3', 5, 5'-tetramethylbenzidine sulfate (TMB), which was generated by HRP in the presence of H2O2. This method exhibited ultrahigh sensitivity to tDNA with detection limit of 0.4 fM. Furthermore, the biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences.

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

    PubMed

    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.

  5. DNA sequence similarity recognition by hybridization to short oligomers

    DOEpatents

    Milosavljevic, Aleksandar

    1999-01-01

    Methods are disclosed for the comparison of nucleic acid sequences. Data is generated by hybridizing sets of oligomers with target nucleic acids. The data thus generated is manipulated simultaneously with respect to both (i) matching between oligomers and (ii) matching between oligomers and putative reference sequences available in databases. Using data compression methods to manipulate this mutual information, sequences for the target can be constructed.

  6. Electrostatics of DNA nucleotide-carbon nanotube hybrids evaluated from QM:MM simulations.

    PubMed

    Chehel Amirani, Morteza; Tang, Tian

    2015-12-14

    Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7,0) were considered. The types of nucleotides and CNTs were both found to play important roles in the electrostatic potential and charge transfer of the hybrid. At the same distance from the CNT axis, the electrostatic potential for the nucleotide-(4,4) CNT hybrids was found to be stronger compared with that for the nucleotide-(7,0) CNT hybrids. Higher electric charge was also shown to be transferred from the DNA nucleotides to the (7,0) CNT compared with the (4,4) CNT. These results correlate with the previous finding that the nucleotides bound more tightly to the (7,0) CNT compared with the (4,4) CNT. PMID:26542447

  7. Broken Optical Symmetry in DNA-SWNT Hybrids: Spectroscopic Signaling of the Helical Wrap

    NASA Astrophysics Data System (ADS)

    Rotkin, Slava V.

    2009-03-01

    Functionalizing single-stranded DNA on a single-wall carbon nanotube (SWNT) has allowed isolating individual tubes, making them soluble, and separating SWNTs according to their chirality. Such strong technological impact motivated our study of the optical properties of the DNA-SWNT hybrids, commonly used now for the solution-based fabrication and experiments. The helicity of the DNA wrap may interfere with the intrinsic Hamiltonian of the SWNT and result in bandstructure modulation. Our modeling predicts a symmetry lowering in the hybrid due to the Coulomb potential of the regular helical wrap of the ionized backbone of the ssDNA, followed by the qualitative changes in the cross- or circularly polarized SWNT absorption spectrum (with no or little change in the parallel polarization). In particular, we predict the appearance of a new peak in the cross-polarized absorption of the ssDNA-SWNT at a frequency lower than that of all allowed transitions in the bare tube. Such effect can be used for optical identification of the wrap at sufficient ssDNA coverage. Wrap signaling happens also via another optical effect, a strong circular dichroism even in the complex with an achiral SWNT, and even at the frequencies where ss-DNA has no absorption features at all. Symmetry of the wrap is central to determine such a circular dichroism of the hybrid. Having in mind that the exact geometry of a DNA wrap for an arbitrary tube is not precisely known yet, we put forward a general model capable of tracking optical effects, varying the parameters of the wrap and/or tube diameter. For various ssDNA backbone helical angles and for various tubes we predict different absorption spectra, though a general qualitative feature of the helical symmetry breaking, the appearance of new van Hove singularities and circular dichroism, must be present.

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

    PubMed

    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. PMID:27498231

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

    PubMed

    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.

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

    PubMed

    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.

  11. 1-ethynylpyrene-modified guanine and cytosine as optical labels for DNA hybridization.

    PubMed

    Wagner, Clemens; Rist, Manuela; Mayer-Enthart, Elke; Wagenknecht, Hans-Achim

    2005-06-01

    1-ethynylpyrene shows remarkable absorption changes upon DNA hybridization when it is covalently attached to the 8-position of guanine. An absorption band at approximately 420 nm is only present in the duplex, exhibits thermal melting behaviour and provides the basis for a molecular beacon together with 1-ethynylpyrene-modified cytosine.

  12. Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

    PubMed

    Kavanagh, Paul; Leech, Dónal

    2006-04-15

    The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach. PMID:16615783

  13. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  14. Use of synthetic oligonucleotides for genomic DNA dot hybridization to split the DQw3 haplotype.

    PubMed Central

    Martell, M; Le Gall, I; Millasseau, P; Dausset, J; Cohen, D

    1988-01-01

    Comparison of two different HLA-DQ beta gene sequences from two DR4 individuals, probably corresponding to DQw3.2 (DQR4) and DQw3.1 (DQR5) specificities, has shown several nucleotide variations. Eight oligonucleotides (24 bases long), derived from these polymorphic areas, have been synthesized. Each oligonucleotide was hybridized to BamHI-digested DNA samples from eight families with HLA-DR4 individuals. Four polymorphic BamHI fragments were detected. Two of eight oligonucleotides gave a single signal (8.9 kilobases) on DQw3.2-positive haplotypes. We used one of these oligonucleotides in a genomic DNA dot hybridization and detected a hybridization signal only in DQw3.2-positive individuals. A very simple test like this allows the screening of a large population sample within a very short period. Images PMID:2895927

  15. [Three cases of vulvar bowenoid papulosis: the localization of HPV DNA by in situ hybridization].

    PubMed

    Kioka, H; Nagai, N; Tanioka, Y; Fujii, T; Katsube, Y; Egawa, K; Fujiwara, A

    1989-09-01

    Cytological, histological, and molecular biological studies were conducted in 3 cases of vulvar Bowenoid papulosis, using biotinylated HPV DNA probes by in situ hybridization. 1) Cytological findings showed dyskaryotic cells that revealed hyperchromatism with a coarse granular pattern, and a high N/C ratio was observed among the dyskeratotic cells. 2) In 2 cases of Bowenoid papulosis lesions, HPV 16 DNA was detected in the nucleus of the dysplastic cells. 3) In one case of Bowenoid papulosis, a complicated carcinoma in situ of the uterine cervix was observed, and the HPV 16 DNA was found to be positive in both the vulva and cervix. PMID:2550688

  16. Ultrasensitive Detection of DNA Hybridization Using Carbon Nanotube Field-Effect Transistors

    NASA Astrophysics Data System (ADS)

    Maehashi, Kenzo; Matsumoto, Kazuhiko; Kerman, Kagan; Takamura, Yuzuru; Tamiya, Eiichi

    2004-12-01

    We have sensitively detected DNA hybridization using carbon nanotube field-effect transistors (CNTFETs) in real time. Amino modified peptide nucleic acid (PNA) oligonucleotides at 5' end were covalently immobilized onto the Au surface of the back gate. For 11-mer PNA oligonucletide probe, full-complementary DNA with concentration as low as 6.8 fM solution could be effectively detected. Our CNTFET-based biochip is a promising candidate for the development of an integrated, high-throughput, multiplexed DNA biosensor for medical, forensic and environmental diagnostics.

  17. Shape Analysis of DNA-Au Hybrid Particles by Analytical Ultracentrifugation.

    PubMed

    Urban, Maximilan J; Holder, Isabelle T; Schmid, Marius; Fernandez Espin, Vanesa; Garcia de la Torre, Jose; Hartig, Jörg S; Cölfen, Helmut

    2016-08-23

    Current developments in nanotechnology have increased the demand for nanocrystal assemblies with well-defined shapes and tunable sizes. DNA is a particularly well-suited building block in nanoscale assemblies because of its scalable sizes, conformational variability, and convenient self-assembly capabilities via base pairing. In hybrid materials, gold nanoparticles (AuNPs) can be assembled into nanoparticle structures with programmable interparticle distances by applying appropriate DNA sequences. However, the development of stoichiometrically defined DNA/NP structures is still challenging since product mixtures are frequently obtained and their purification and characterization is the rate-limiting step in the development of DNA-NP hybrid assemblies. Improvements in nanostructure fractionation and characterization techniques offer great potential for nanotechnology applications in general. This study reports the application of analytical ultracentrifugation (AUC) for the characterization of anisotropic DNA-linked metal-crystal assemblies. On the basis of transmission electron microscopy data and the DNA primary sequence, hydrodynamic bead models are set up for the interpretation of the measured frictional ratios and sedimentation coefficients. We demonstrate that the presence of single DNA strands on particle surfaces as well as the shape factors of multiparticle structures in mixtures can be quantitatively described by AUC. This study will significantly broaden the possibilities to analyze mixtures of shape-anisotropic nanoparticle assemblies. By establishing insights into the analysis of nanostructure mixtures based on fundamental principles of sedimentation, a wide range of potential applications in basic research and industry become accessible. PMID:27459174

  18. Layered zirconium phosphonate with inorganic-organic hybrid structure: Preparation and its assembly with DNA

    NASA Astrophysics Data System (ADS)

    Liu, Li-Min; Lu, Guo-Yuan; Jiang, Li-Ping; Zhu, Jun-Jie

    2014-07-01

    An aminoethoxy-functionalized zirconium phosphonate (Zr(O3POCH2CH2NH2)2·3H2O), abbreviated as ZrRP (R=OCH2CH2NH2), with layered structure has been synthesized. This layered compound possesses the characteristic of inorganic-organic hybrid, due to the covalently linked aminoethoxy in the host layer. The anion exchanged property of this zirconium phosphonate is suitable for the direct intercalation of negatively charged DNA, which is different from these reported zirconium phosphates or zirconium phosphonates. As a precursor, this prepared zirconium phosphonate was utilized to fabricate a novel DNA/ZrRP binary hybrid via a delamination-reassembly procedure. The release behavior of DNA from the DNA/ZrRP composite was investigated at different medium pH, because the combination between zirconium phosphonate sheets and DNA was pH-dependent sensitively. Moreover, the helical conformation of DNA was almost retained after the intercalation and release process. These properties of the DNA/ZrRP composite suggested the potential application of layered zirconium phosphonate as a non-viral vector in gene delivery.

  19. An impedimetric study of DNA hybridization on paper-supported inkjet-printed gold electrodes

    NASA Astrophysics Data System (ADS)

    Ihalainen, Petri; Pettersson, Fredrik; Pesonen, Markus; Viitala, Tapani; Määttänen, Anni; Österbacka, Ronald; Peltonen, Jouko

    2014-03-01

    In this study, two different supramolecular recognition architectures for impedimetric detection of DNA hybridization have been formed on disposable paper-supported inkjet-printed gold electrodes. The gold electrodes were fabricated using a gold nanoparticle based ink. The first recognition architecture consists of subsequent layers of biotinylated self-assembly monolayer (SAM), streptavidin and biotinylated DNA probe. The other recognition architecture is constructed by immobilization of thiol-functionalized DNA probe (HS-DNA) and subsequent backfill with 11-mercapto-1-undecanol (MUOH) SAM. The binding capacity and selectivity of the recognition architectures were examined by surface plasmon resonance (SPR) measurements. SPR results showed that the HS-DNA/MUOH system had a higher binding capacity for the complementary DNA target. Electrochemical impedance spectroscopy (EIS) measurements showed that the hybridization can be detected with impedimetric spectroscopy in picomol range for both systems. EIS signal indicated a good selectivity for both recognition architectures, whereas SPR showed very high unspecific binding for the HS-DNA/MUOH system. The factors affecting the impedance signal were interpreted in terms of the complexity of the supramolecular architecture. The more complex architecture acts as a less ideal capacitive sensor and the impedance signal is dominated by the resistive elements.

  20. Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization.

    PubMed

    Verhaegent, Monique; Christopoulos, Theodore K

    2002-09-01

    The cDNA for Gaussia luciferase (GLuc), the enzyme responsible for the bioluminescent reaction of the marine copepod Gaussia princeps, has been cloned recently. GLuc (MW = 19 900) catalyzes the oxidative decarboxylation of coelenterazine to produce coelenteramide and light. We report the first quantitative anaytical study of GLuc and examine its potential as a new reporter for DNA hybridization. A plasmid encoding both a biotin acceptor peptide-GLuc fusion protein as well as the enzyme biotin protein ligase (BPL) is engineered by using GLuc cDNA as a starting template. BPL catalyzes the covalent attachment of a single biotin to the fusion protein in vivo. Purification of GLuc is then accomplished by affinity chromatography using immobilized monomeric avidin. Moreover, the in vivo biotinylation enables subsequent complexation of GLuc with streptavidin (SA), thereby avoiding chemical conjugation reactions that are known to inactivate luciferases. Purified GLuc can be detected down to 1 amol with a signal-to-background ratio of 2 and a linear range extending over 5 orders of magnitude. The background luminescence of coelenterazine is the main limiting factor for even higher detectability of GLuc. Furthermore, the GLuc-SA complex is used as a detection reagent in a microtiter well-based DNA hybridization assay. The analytical range extends from 1.6 to 800 pmol/L of target DNA. Biotinylated GLuc produced from 1 L of bacterial culture is sufficient for 150,000 hybridization assays.

  1. Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

    PubMed

    Gong, Ping; Lee, Chi-Ying; Gamble, Lara J; Castner, David G; Grainger, David W

    2006-05-15

    Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats. PMID:16689533

  2. The intrinsic role of nanoconfinement in chemical equilibrium: evidence from DNA hybridization.

    PubMed

    Rubinovich, Leonid; Polak, Micha

    2013-05-01

    Recently we predicted that when a reaction involving a small number of molecules occurs in a nanometric-scale domain entirely segregated from the surrounding media, the nanoconfinement can shift the position of equilibrium toward products via reactant-product reduced mixing. In this Letter, we demonstrate how most-recently reported single-molecule fluorescence measurements of partial hybridization of ssDNA confined within nanofabricated chambers provide the first experimental confirmation of this entropic nanoconfinement effect. Thus, focusing separately on each occupancy-specific equilibrium constant, quantitatively reveals extra stabilization of the product upon decreasing the chamber occupancy or size. Namely, the DNA hybridization under nanoconfined conditions is significantly favored over the identical reaction occurring in bulk media with the same reactant concentrations. This effect, now directly verified for DNA, can be relevant to actual biological processes, as well as to diverse reactions occurring within molecular capsules, nanotubes, and other functional nanospaces.

  3. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function

    PubMed Central

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-01-01

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition–fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates “bifacial polymer nucleic acids” (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure–function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  4. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    PubMed

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  5. DNA breakage detection-fluorescence in situ hybridization in buccal cells

    PubMed Central

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

    2012-01-01

    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. PMID:23361245

  6. Synthesis of nucleobase-functionalized carbon nanotubes and their hybridization with single-stranded DNA.

    PubMed

    Hwu, Jih Ru; Kapoor, Mohit; Li, Rou-Ying; Lin, Yung-Chieh; Horng, Jia-Cherng; Tsay, Shwu-Chen

    2014-12-01

    For the first time ssDNA (25-aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single-walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole-(ethylene glycol)-dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π-π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.

  7. Label-free detection of DNA hybridization at a liquid|liquid interface.

    PubMed

    Vagin, Mikhail Yu; Trashin, Stanislav A; Karyakin, Arkady A; Mascini, Marco

    2008-02-15

    A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of ion-transfer resistance. The detection limit was of 10-8 M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters.

  8. A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

    PubMed Central

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

    2014-01-01

    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. PMID:25285529

  9. Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus.

    PubMed

    Matyášek, Roman; Dobešová, Eva; Húska, Dalibor; Ježková, Ivana; Soltis, Pamela S; Soltis, Douglas E; Kovařík, Aleš

    2016-02-01

    Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids. PMID:26711705

  10. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    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.

  11. Electronic hybridization detection in microarray format and DNA genotyping

    NASA Astrophysics Data System (ADS)

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-02-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

  12. DNA Hybridization-Mediated Liposome Fusion at the Aqueous Liquid Crystal Interface

    PubMed Central

    Noonan, Patrick S.; Mohan, Praveena; Goodwin, Andrew P.

    2014-01-01

    The prominence of receptor-mediated bilayer fusion in cellular biology motivates development of biomimetic strategies for studying fusogenic mechanisms. An approach is reported here for monitoring receptor-mediated fusion that exploits the unique physical and optical properties of liquid crystals (LC). PEG-functionalized lipids are used to create an interfacial environment capable of inhibiting spontaneous liposome fusion with an aqueous/LC interface. Then, DNA hybridization between oligonucleotides within bulk phase liposomes and a PEG-lipid monolayer at an aqueous/LC interface is exploited to induce receptor-mediated liposome fusion. These hybridization events induce strain within the liposome bilayer, promote lipid mixing with the LC interface, and consequently create an interfacial environment favoring re-orientation of the LC to a homeotropic (perpendicular) state. Furthermore, the bi-functionality of aptamers is exploited to modulate DNA hybridization-mediated liposome fusion by regulating the availability of the appropriate ligand (i.e., thrombin). Here, a LC-based approach for monitoring receptor (i.e., DNA hybridization)-mediated liposome fusion is demonstrated, liposome properties that dictate fusion dynamics are explored, and an example of how this approach may be used in a biosensing scheme is provided. PMID:25506314

  13. Functionalized ensembles of nanoelectrodes as affinity biosensors for DNA hybridization detection.

    PubMed

    Silvestrini, Morena; Fruk, Ljiljana; Ugo, Paolo

    2013-02-15

    A novel electrochemical biosensor for DNA hybridization detection based on nanoelectrode ensembles (NEEs) is presented. NEEs are prepared by electroless deposition of gold into the pores of a templating track-etched polycarbonate (PC) membrane. The wide surface of the templating membrane surrounding the nanoelectrodes is exploited to bind the capture DNA probes via amide coupling with the carboxylic groups present on the PC surface. The probes are then hybridized with the complementary target labelled with glucose oxidase (GO(x)). The occurrence of the hybridization event is detected by adding, to the supporting electrolyte, excess glucose as the substrate and the (ferrocenylmethyl) trimethylammonium cation (FA(+)) as suitable redox mediator. In the case of positive hybridization, an electrocatalytic current is detected. In the proposed sensor, the biorecognition event and signal transduction occur in different but neighbouring sites, i.e., the PC surface and the nanoelectrodes, respectively; these sites are separated albeit in close proximity on a nanometer scale. Finally, the possibility to activate the PC surface by treatment with permanganate is demonstrated and the analytical performances of biosensors prepared with KMnO(4)-treated NEEs and native NEEs are compared and critically evaluated. The proposed biosensor displays high selectivity and sensitivity, with the capability to detect few picomoles of target DNA.

  14. Direct immobilization and hybridization of DNA on group III nitride semiconductors

    NASA Astrophysics Data System (ADS)

    Xu, Xiaobin; Jindal, Vibhu; Shahedipour-Sandvik, Fatemeh; Bergkvist, Magnus; Cady, Nathaniel C.

    2009-03-01

    A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.

  15. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    PubMed

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

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

    PubMed

    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.

  17. Nanomechanics of the formation of DNA self-assembled monolayers and hybridization on microcantilevers.

    PubMed

    Alvarez, M; Carrascosa, L G; Moreno, M; Calle, A; Zaballos, A; Lechuga, L M; Martínez-A, C; Tamayo, J

    2004-10-26

    Biomolecular interactions over the surface of a microcantilever can produce its bending motion via changes of the surface stress, which is referred to nanomechanical response. Here, we have studied the interaction forces responsible for the bending motion during the formation of a self-assembled monolayer of thiolated 27-mer single-stranded DNA on the gold-coated side of a microcantilever and during the subsequent hybridization with the complementary nucleic acid. The immobilization of the single-stranded DNA probe gives a mean surface stress of 25 mN/m and a mean bending of 23 nm for microcantilevers with a length and thickness of about 200 microm and 0.8 microm, respectively. The hybridization with the complementary sequence could not be inferred from the nanomechanical response. The nanomechanical response was compared with data from well-established techniques such as surface plasmon resonance and radiolabeling, to determine the surface coverage and study the intermolecular forces between neighboring DNA molecules anchored to the microcantilever surface. From both techniques, an immobilization surface density of 3 x 10(12) molecules/cm(2) and a hybridization efficiency of 40% were determined. More importantly, label-free hybridization was clearly detected in the same conditions with a conventional sensor based on surface plasmon resonance. The results imply that the nanomechanical signal during the immobilization process arises mainly from the covalent attachment to the gold surface, and the interchain interactions between neighboring DNA molecules are weak, producing an undetectable surface stress. We conclude that detection of nucleic acid hybridization with nanomechanical sensors requires reference cantilevers to remove nonspecific signals, more sensitive microcantilever geometries, and immobilization chemistries specially addressed to enhance the surface stress variations.

  18. Random amplified polymorphic DNA analysis, genome size, and genomic in situ hybridization of triploid viviparous onions

    PubMed

    Puizina; Javornik; Bohanec; Schweizer; Maluszynska; Papes

    1999-12-01

    Triploid viviparous onions (Allium cepa L. var. viviparum Metzg. (ALEF.), auct.), (2n = 3x = 24), are known in some countries only as a rare relic crop, while in other parts of the world they are still traditionally or even commercially cultivated. Results indicating an identical random amplified polymorphic DNA (RAPD) banding pattern and the same DNA content (2C = 43.4 pg) establish the high genetic similarity and the unique origin of the Croatian clone Ljutika and the Indian clone Pran. In order to determine the parental Allium species of these natural triploid hybrids, genomic fluorescent in situ hybridization (GISH) was applied. Biotinylated genomic DNAs from six diploid Allium species (A. cepa L., A. fistulosum L., A. roylei Stearn, A. vavilovii M. Pop. et Vved., A. galanthum Kar. et Kir., A. oschaninii O. Fedtsch.) were used as probes in this study. While probes obtained from genomic DNA of A. cepa, A. vavilovii, and A. roylei hybridized to somatic chromosomes of Ljutika probes from A. fistulosum, A. galanthum, and A. oschaninii did not. The DNA probes of A. cepa and A. roylei each completely or predominantly labelled one genome (eight chromosomes). A few chromosomes, the markers of the triploid karyotype, were not completely labelled by any probe applied. Our GISH results indicate that triploid viviparous onions might possess a complex triparental genome organization. PMID:10659789

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

    PubMed

    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.

  20. Comprehensive evaluation of two HLA-B17 monoclonal antibodies for flow cytometry-based HLA-B57/B58 screening prior to abacavir prescription.

    PubMed

    Stevens, R; Coates, E; Street, J; Cook, E; Darke, C

    2013-08-01

    Hypersensitivity reactions to the drug abacavir, used to treat HIV/AIDS patients, is associated with possession of HLA-B*57:01. We have carefully assessed two commercially available HLA-B57/B58 murine monoclonal antibodies [0196HA and BIH0243 (One Lambda Inc.)] in a simple flow cytometry-based assay. The evaluation involved tests on 228 reference and random samples covering 91% of all WHO recognized HLA-A, B and C specificities. These involved donors with six different HLA-B*57 alleles and included 19 examples of B*57:01. Both antibodies unambiguously detected B57, but there were small difference in their reactivity against B57-positive non-B*57:01 samples. Importantly, there was no reactivity against B57/B58-negative samples. The possible amino acid motifs involved in the reactivity of these antibodies with B57/B58 were delineated. Thus, HLA-B57/B58, normally present in <10% of patients, can be easily recognized using these two antibodies and further tested by a DNA-based typing method to identify B*57:01.

  1. Quantitative High-Resolution Sensing of DNA Hybridization Using Magnetic Tweezers with Evanescent Illumination

    PubMed Central

    Oliver, Piercen M.; Park, Jin Seon; Vezenov, Dmitri

    2012-01-01

    We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescent image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force-distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy. PMID:21103547

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

    PubMed

    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.

  3. Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

    PubMed

    Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

    2016-08-24

    A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence.

  4. An upconversion fluorescent resonant energy transfer biosensor for hepatitis B virus (HBV) DNA hybridization detection.

    PubMed

    Zhu, Hao; Lu, Feng; Wu, Xing-Cai; Zhu, Jun-Jie

    2015-11-21

    A novel fluorescent resonant energy transfer (FRET) biosensor was fabricated for the detection of hepatitis B virus (HBV) DNA using poly(ethylenimine) (PEI) modified upconversion nanoparticles (NH2-UCNPs) as energy donor and gold nanoparticles (Au NPs) as acceptor. The PEI modified upconversion nanoparticles were prepared directly with a simple one-pot hydrothermal method, which provides high quality amino-group functionalized UCNPs with uniform morphology and strong upconversion luminescence. Two single-stranded DNA strands, which were partially complementary to each other, were then conjugated with NH2-UCNPs and Au NPs. When DNA conjugated NH2-UCNPs and Au NPs are mixed together, the hybridization between complementary DNA sequences on UCNPs and Au NPs will lead to the quenching of the upconversion luminescence due to the FRET process. Meanwhile, upon the addition of target DNA, Au NPs will leave the surface of the UCNPs and the upconversion luminescence can be restored because of the formation of the more stable double-stranded DNA on the UCNPs. The sensor we fabricated here for target DNA detection shows good sensitivity and high selectivity, which has the potential for clinical applications in the analysis of HBV and other DNA sequences. PMID:26421323

  5. The effects of multiple probes on the hybridization of target DNA on surfaces

    NASA Astrophysics Data System (ADS)

    Welling, Ryan C.; Knotts, Thomas A.

    2015-01-01

    DNA microarrays have disruptive potential in many fields including genetics and medicine, but the technology has yet to find widespread clinical use due to poor reliability. Microarrays work on the principle of hybridization and can only be as dependable as this process is reliable. As such, a significant amount of theoretical research has been done to understand hybridization on surfaces on the molecular level. Previous simulations of a target strand with a single, surface-tethered probe molecule have yielded valuable insights, but such is an ideal system and little is known about the effects of multiple probes—a situation that more closely approximates the real system. This work uses molecular simulation to determine the specific differences in duplex stability between one, three, six, and nine tethered probes on a surface. The results show that it is more difficult for a single target to hybridize to a probe as the number of probes on the surface increases due to crowding effects; however, once hybridized, the duplex is more stable than when fewer probes are present. The data also indicate that hybridization of a target to a probe on the face of a group of probes is more stable than hybridization to probes at the edge or center locations. Taken as a whole, the results offer new insights into the cause of the poor reproducibility exhibited by microarrays.

  6. Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

    PubMed Central

    Lee, Chung-Te; Amaro, Carmen; Sanjuán, Eva; Hor, Lien-I

    2005-01-01

    Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains. PMID:16151155

  7. Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium.

    PubMed Central

    Poppe, C; Curtiss, R; Gulig, P A; Gyles, C L

    1989-01-01

    Plasmid DNA of 68 strains of Salmonella that belonged to 18 serovars and exhibited 48 different plasmid profiles was examined for hybridization with a 32P-labelled DNA probe which consisted of a 3750 base pairs (bp) HindIII-HindIII fragment derived from the virulence region of the 60 megadalton (Mdal) plasmid of Salmonella typhimurium. The 32 Mdal plasmid of S. cholerae-suis, the 50 Mdal plasmid of S. dublin, the 36 Mdal plasmid of S. enteritidis, the 60 Mdal plasmid of S. gallinarum, the 60 Mdal plasmid of S. pullorum, and the 60 Mdal plasmid of S. typhimurium, plasmids that have been associated with virulence, all hybridized with the probe. Digestion of plasmid DNA of these strains with PvuII and hybridization with the probe revealed that the plasmids of strains of all six serovars contained fragments of approximately 2520 and 1520 bp that hybridized with the probe. Similarly, hybridization with BglI digests of DNA of the virulence-associated plasmids of strains of these six serovars showed that all six plasmids contained a fragment of approximately 3690 bp that hybridized with the probe. No other plasmids of these strains nor any plasmids of 12 other Salmonella serovars hybridized with the probe. Chromosomal DNA did not hybridize with the probe. The 60 Mdal plasmids of S. gallinarum and S. pullorum showed similar digestion patterns with restriction endonucleases BglI, BglII and PvuII. Images Fig. 2. Fig. 3. Fig. 4. PMID:2686827

  8. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

    PubMed Central

    Wahl, G M; Stern, M; Stark, G R

    1979-01-01

    We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly. Images PMID:291033

  9. Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces.

    PubMed Central

    Kuritza, A P; Salyers, A A

    1985-01-01

    pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4083890

  10. Electrostatics of DNA nucleotide-carbon nanotube hybrids evaluated from QM:MM simulations

    NASA Astrophysics Data System (ADS)

    Chehel Amirani, Morteza; Tang, Tian

    2015-11-01

    Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7,0) were considered. The types of nucleotides and CNTs were both found to play important roles in the electrostatic potential and charge transfer of the hybrid. At the same distance from the CNT axis, the electrostatic potential for the nucleotide-(4,4) CNT hybrids was found to be stronger compared with that for the nucleotide-(7,0) CNT hybrids. Higher electric charge was also shown to be transferred from the DNA nucleotides to the (7,0) CNT compared with the (4,4) CNT. These results correlate with the previous finding that the nucleotides bound more tightly to the (7,0) CNT compared with the (4,4) CNT.Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7

  11. Improvement of DNA recognition through molecular imprinting: hybrid oligomer imprinted polymeric nanoparticles (oligoMIP NPs).

    PubMed

    Brahmbhatt, H; Poma, A; Pendergraff, H M; Watts, J K; Turner, N W

    2016-02-01

    High affinity and specific binding are cardinal properties of nucleic acids in relation to their biological function and their role in biotechnology. To this end, structural preorganization of oligonucleotides can significantly improve their binding performance, and numerous examples of this can be found in Nature as well as in artificial systems. Here we describe the production and characterization of hybrid DNA-polymer nanoparticles (oligoMIP NPs) as a system in which we have preorganized the oligonucleotide binding by molecular imprinting technology. Molecularly imprinted polymers (MIPs) are cost-effective "smart" polymeric materials capable of antibody-like detection, but characterized by superior robustness and the ability to work in extreme environmental conditions. Especially in the nanoparticle format, MIPs are dubbed as one of the most suitable alternatives to biological antibodies due to their selective molecular recognition properties, improved binding kinetics as well as size and dispersibility. Nonetheless, there have been very few attempts at DNA imprinting in the past due to structural complexity associated with these templates. By introducing modified thymine bases into the oligonucleotide sequences, which allow establishing covalent bonds between the DNA and the polymer, we demonstrate that such hybrid oligoMIP NPs specifically recognize their target DNA, and that the unique strategy of incorporating the complementary DNA strands as "preorganized selective monomers" improves the recognition properties without affecting the NPs physical properties such as size, shape or dispersibility. PMID:26509192

  12. Modelling cross-hybridization on phylogenetic DNA microarrays increases the detection power of closely related species.

    PubMed

    Engelmann, Julia C; Rahmann, Sven; Wolf, Matthias; Schultz, Jörg; Fritzilas, Epameinondas; Kneitz, Susanne; Dandekar, Thomas; Müller, Tobias

    2009-01-01

    DNA microarrays are a popular technique for the detection of microorganisms. Several approaches using specific oligomers targeting one or a few marker genes for each species have been proposed. Data analysis is usually limited to call a species present when its oligomer exceeds a certain intensity threshold. While this strategy works reasonably well for distantly related species, it does not work well for very closely related species: Cross-hybridization of nontarget DNA prevents a simple identification based on signal intensity. The majority of species of the same genus has a sequence similarity of over 90%. For biodiversity studies down to the species level, it is therefore important to increase the detection power of closely related species. We propose a simple, cost-effective and robust approach for biodiversity studies using DNA microarray technology and demonstrate it on scenedesmacean green algae. The internal transcribed spacer 2 (ITS2) rDNA sequence was chosen as marker because it is suitable to distinguish all eukaryotic species even though parts of it are virtually identical in closely related species. We show that by modelling hybridization behaviour with a matrix algebra approach, we are able to identify closely related species that cannot be distinguished with a threshold on signal intensity. Thus this proof-of-concept study shows that by adding a simple and robust data analysis step to the evaluation of DNA microarrays, species detection can be significantly improved for closely related species with a high sequence similarity.

  13. Helicoidal Fields and Spin Polarized Currents in Carbon Nanotube-DNA Hybrids

    NASA Astrophysics Data System (ADS)

    Diniz, G. S.; Latgé, A.; Ulloa, S. E.

    2012-03-01

    We report on theoretical studies of electronic transport in the archetypical molecular hybrid formed by DNA wrapped around single-walled carbon nanotubes (CNTs). Using a Green’s function formalism in a π-orbital tight-binding representation, we investigate the role that spin-orbit interactions play on the CNT in the case of the helicoidal electric field induced by the polar nature of the adsorbed DNA molecule. We find that spin polarization of the current can take place in the absence of magnetic fields, depending strongly on the direction of the wrapping and length of the helicoidal field. These findings open new routes for using CNTs in spintronic devices.

  14. Localization of cytomegalovirus DNA in plastic-embedded sections by in situ hybridization. A methodologic study.

    PubMed Central

    Cao, M.; Beckstead, J. H.

    1989-01-01

    The use of in situ hybridization for the identification of specific nucleic acid sequences in tissue sections has the potential for broad application in pathology. Although this technique has been successfully applied to routine paraffin sections, there have been few studies of the application of in situ hybridization to plastic-embedded tissue sections. The authors adapted techniques developed for paraffin sections to take advantage of the potential for improved morphology and more precise localization inherent in the plastic sections. A commercially available biotinylated DNA probe specific for the cytomegalovirus to develop a practical method for detection of nucleic acid sequences in plastic-embedded tissues was used. Using plastic sections, cytomegalovirus DNA sequences could readily be identified with precise localization of the virus and superb histology. Images Figure 2 Figure 3 Figure 1 Figure 4 PMID:2537020

  15. A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

    PubMed

    Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

    2008-11-01

    A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

  16. Rapid Determination of RNA Accessible Sites by Surface Plasmon Resonance Detection of Hybridization to DNA arrays

    PubMed Central

    Mandir, Joshua B.; Lockett, Matthew R.; Phillips, Margaret F.; Allawi, Hatim T.; Lyamichev, Victor I.; Smith, Lloyd M.

    2009-01-01

    RNA accessible sites are the regions in an RNA molecule, which are available for hybridization with complementary DNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of ~75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array comprised of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner. PMID:19874056

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

    SciTech Connect

    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.

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

    PubMed

    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.

  19. Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

    2014-09-01

    The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide

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

    DOEpatents

    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. Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

    SciTech Connect

    Pallan, Pradeep S.; Egli, Martin

    2009-06-17

    Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme's interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg{sup 2+} at the active site. A subset of amino acids engaged in contacts to RNA 2{prime}-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme's interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.

  2. Characterization of honeybee (Apis mellifera L.) chromosomes using repetitive DNA probes and fluorescence in situ hybridization.

    PubMed

    Beye, M; Moritz, R F

    1995-01-01

    Two different repetitive DNA probes of Apis mellifera and ribosomal DNA from Drosophila melanogaster were used to characterize the chromosomal set of the honeybee (n = 16). The probes were hybridized to chromosome preparations of haploid testis tissue from drone larvae using fluorescence in situ hybridization (FISH). The honeybee probes hybridized to the telomeric (Alu I family) and centromeric region (Ava I family) of most chromosomes. The rDNA probe labeled two chromosomes only. Combination of the three probes yielded labeled patterns allowing us to identify each chromosome of the honeybee individually. This is the first report of an unambiguous identification of the chromosomal set of the honeybee, since classical banding techniques failed to yield clear patterns for identification. The consensus sequence of the centromeric reiterated probe (Ava I family) has a length of about 550 nucleotides and shows no homology to other known sequences. However, the structural organization of a 130-nucleotides long motif forming the unusually homogeneous 550 nucleotides repeat is similar to those found in mammals' repetitive DNAs.

  3. Detection of Salmonella by using the colorimetric DNA/rRNA sandwich hybridization in microtiter wells.

    PubMed

    Namimatsu, T; Tsuna, M; Imai, Y; Futo, S; Mitsuse, S; Sakano, T; Sato, S

    2000-06-01

    A rapid and readily available DNA probe kit was developed for the detection of Salmonella spp. This kit utilized the colorimetric DNA/rRNA sandwich hybridization method in microtiter wells. Within 3 hr Salmonella spp. in selective enrichment broth cultures were detected by the DNA probe kit. The kit effectively identified all of 187 strains of Salmonella tested and yielded no false-positive reactions in the examination of 674 pure cultures of non-salmonellae. The DNA probe kit could detect 10(5) cfu/ml in pure culture. A total of 379 naturally contaminated samples (raw chicken meat, liquid egg, animal feeds, poultry feces and frozen foods) were tested, both by the standard culture method and the DNA probe kit. The 169 of these samples were culture positive and 210 were culture negative. The sensitivity of the DNA probe kit was 98.2% (166/169) and the specificity was 99.5% (209/210). These results show that the DNA probe kit is a useful tool to examine a large number of various samples for contamination by Salmonella spp. in food and livestock industry. PMID:10907688

  4. DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity.

    PubMed

    Xu, Zhenning; Yin, Huanshun; Han, Yunxiang; Zhou, Yunlei; Ai, Shiyun

    2014-06-01

    In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 uni tmL(-1). Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism. PMID:24856396

  5. In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions.

    PubMed

    Syrjänen, S M; Syrjänen, K J; Happonen, R P; Lamberg, M A

    1987-01-01

    A series of 144 surgically treated benign oral mucosal lesions were analysed using an in situ DNA hybridization technique with 35S-labeled human papillomavirus (HPV) DNA probes to demonstrate the DNA of HPV types 6, 11, 13, and 16, in routinely processed, paraffin-embedded biopsy specimens. These lesions and an additional 62 benign oral mucosal biopsy specimens (total, 206 specimens) were also assessed by the indirect immunoperoxidase (IP-PAP) technique to detect the expression of HPV structural proteins (viral antigens). A total of 54/206 (26.2%) lesions were observed to express HPV antigens, being found in 45/92 (48.9%) of the squamous cell papillomas/condylomas, in 1/54 fibrous hyperplasias, in 1/8 true fibromas, and in 7/8 (87.5%) of the focal epithelial hyperplasia (FEH) lesions. Of the HPV DNA-positive lesions, 15/45 (33.3%) expressed HPV antigens, the expression not being related to any particular HPV type. HPV DNA sequences were found in 45/144 (31.3%) of the lesions. HPV DNA was present with the highest frequency in FEH (83.3%), followed by the papilloma/condyloma group (33.8%), papillary hyperplasia (28.6%), fibrous hyperplasia (24.4%), and true fibromas (14.3%). The most frequent HPV type was HPV 11, representing 37.8% of the DNA-positive lesions. HPV 13 DNA, previously regarded as specific to FEH, was disclosed as a single HPV type in seven cases, and as a double infection by HPV 11 and 13 in an additional three cases, including all five morphologically distinct entities. Noteworthy is the discovery of the high-risk HPV type 16 DNA in 17.8% of the DNA-positive lesions, four papilloma/condyloma lesions, three fibrous hyperplasias, and one FEH.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

    2016-01-01

    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. PMID:27534818

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27534818

  9. Rotating rod renewable microcolumns for automated, solid-phase DNA hybridization studies.

    PubMed

    Bruckner-Lea, C J; Stottlemyre, M S; Holman, D A; Grate, J W; Brockman, F J; Chandler, D P

    2000-09-01

    The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acid hybridization in an automated sequential injection system is described. The flow cell included a stepper motor-driven rotating rod with the working end cut to a 45 degrees angle. In one position, the end of the rod prevented passage of microbeads while allowing fluid flow; rotation of the rod by 180 degrees releases the beads. This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization. Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately postcolumn during all column perfusion and elution steps using a flow-through fluorescence detector. Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization. Perfusion of the column with complementary oligonucleotide (200 microL, 10 nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than 1 s and a total sample perfusion time of 40 s. The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated. PMID:10994975

  10. Rotating rod renewable microcolumns for automated, solid-phase DNA hybridization studies.

    PubMed

    Bruckner-Lea, C J; Stottlemyre, M S; Holman, D A; Grate, J W; Brockman, F J; Chandler, D P

    2000-09-01

    The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acid hybridization in an automated sequential injection system is described. The flow cell included a stepper motor-driven rotating rod with the working end cut to a 45 degrees angle. In one position, the end of the rod prevented passage of microbeads while allowing fluid flow; rotation of the rod by 180 degrees releases the beads. This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization. Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately postcolumn during all column perfusion and elution steps using a flow-through fluorescence detector. Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization. Perfusion of the column with complementary oligonucleotide (200 microL, 10 nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than 1 s and a total sample perfusion time of 40 s. The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated.

  11. Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation.

    PubMed

    Zhang, Qingzhu; Wang, Dong; Lang, Zhaobo; He, Li; Yang, Lan; Zeng, Liang; Li, Yanqiang; Zhao, Cheng; Huang, Huan; Zhang, Heng; Zhang, Huiming; Zhu, Jian-Kang

    2016-07-19

    DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. PMID:27382183

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

    SciTech Connect

    Yasuda, Jun; Sekiya, Takao; Navarro, J.M.

    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.

  13. Development of an electrochemical biosensor methods based on acrylic microsphere for the determination of Arowana DNA hybridization

    NASA Astrophysics Data System (ADS)

    Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Chiang, Chew Poh

    2015-09-01

    An electrochemical method of Arowana DNA determination based of N-acrylosuccinimide (NAS) modified acrylic microsphere was fabricated. Hydrophobic succinimide functional group containing poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized with a simple one-step photopolymerization pocedure. Aminated DNA probe was covalently bonded to the succinimde functional group of the acrylic microspheres. The hybridization of the immobilized DNA probe with the complementary DNA was determined by the differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a wide linear response range to target DNA is 1.0 × 10-16 and 1.0 × 10-8 M with a lower limit of detection (LOD) of 9.46 × 10-17 M (R2 = 0.99) were calculated. This biosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices.

  14. Thiazole Orange Dimers in DNA: Fluorescent Base Substitutions with Hybridization Readout.

    PubMed

    Berndl, Sina; Dimitrov, Stoichko D; Menacher, Florian; Fiebig, Torsten; Wagenknecht, Hans-Achim

    2016-02-12

    By using (S)-2-amino-1,3-propanediol as a linker, thiazole orange (TO) was incorporated in a dimeric form into DNA. The green fluorescence (λ=530 nm) of the intrastrand TO dimer is quenched, whereas the interstrand TO dimer shows a characteristic redshifted orange emission (λ=585 nm). Steady-state optical spectroscopic methods reveal that the TO dimer fluorescence is independent of the sequential base contexts. Time-resolved pump-probe measurements and excitation spectra reveal the coexistence of conformations, including mainly stacked TO dimers and partially unstacked ones, which yield exciton and excimer contributions to the fluorescence, respectively. The helicity of the DNA framework distorts the excitonic coupling. In particular, the interstrand TO dimer could be regarded as an excitonically interacting base pair with fluorescence readout for DNA hybridization. Finally, the use of this fluorescent readout was representatively demonstrated in molecular beacons.

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

    PubMed

    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. PMID:25723542

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

    PubMed

    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.

  17. Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

    PubMed

    Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

    2015-12-28

    The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

  18. Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

    PubMed

    Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

    2015-01-01

    The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level. PMID:26782491

  19. Coarse-grained simulation study of sequence effects on DNA hybridization in a concentrated environment.

    PubMed

    Markegard, Cade B; Fu, Iris W; Reddy, K Anki; Nguyen, Hung D

    2015-02-01

    A novel coarse-grained model is developed to elucidate thermodynamics and kinetic mechanisms of DNA self-assembly. It accounts for sequence and solvent conditions to capture key experimental results such as sequence-dependent thermal property and salt-dependent persistence length of ssDNA and dsDNA. Moreover, constant-temperature simulations on two single strands of a homogeneous sequence show two main mechanisms of hybridization: a slow slithering mechanism and a one-order faster zippering mechanism. Furthermore, large-scale simulations at a high DNA strand concentration demonstrate that DNA self-assembly is a robust and enthalpically driven process in which the formation of double helices is deciphered to occur via multiple self-assembly pathways including the strand displacement mechanism. However, sequence plays an important role in shifting the majority of one pathway over the others and controlling size distribution of self-assembled aggregates. This study yields a complex picture on the role of sequence on programmable self-assembly and demonstrates a promising simulation tool that is suitable for studies in DNA nanotechnology. PMID:25581253

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

    SciTech Connect

    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.

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

    PubMed

    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.

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

    PubMed

    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. PMID:18528984

  3. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

    SciTech Connect

    Jiang, X.; Estes, M.K.; Metcalf, T.G.; Melnick, J.L

    1986-10-01

    The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

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

    PubMed

    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.

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

    PubMed

    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.

  6. Rotating Rod Renewable Microcolumns for Automated, Solid-Phase DNA Hybridization

    SciTech Connect

    Bruckner-Lea, Cynthia J. ); Stottlemyre, Mark R.; Holman, David A.; Grate, Jay W. ); Brockman, Fred J. ); Chandler, Darrell P.

    1999-12-01

    The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acid analysis in a sequential injection system is described. The flow cell includes a stepper motor-driven rotating rod with the working end cut to a 45 degree angle. In one position, the end of the rod prevents passage of microbeads while allowing fluid flow; rotation of the rod by 180 degrees release the beads. This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence specific nucleic acid hybridization. Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately post-column using a flow-through fluorescence detector, with a detection limit of 40 pM dye concentration at a flow rate of 5 mu l/s. Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization. Perfusion of the column with complementary oligonucleotide (200 mu l, 10nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than a second and a total sample perfusion time of 40 seconds. The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated.

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

    PubMed

    Reddy, M Siva Pratap; Park, Chinho

    2016-01-01

    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. PMID:27572113

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    Reddy, M Siva Pratap; Park, Chinho

    2016-01-01

    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.

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

    PubMed Central

    Reddy, M. Siva Pratap; Park, Chinho

    2016-01-01

    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. PMID:27572113

  11. In situ hybridization analysis of human papillomavirus DNA in oral mucosal lesions.

    PubMed

    Zeuss, M S; Miller, C S; White, D K

    1991-06-01

    Commercial biotinylated DNA probes specific for human papillomavirus (HPV) types 6 and 11; 16 and 18; and 31, 33, and 35 were used for in situ hybridization analysis of 105 oral mucosal specimens from 5 cases of verruca vulgaris, 15 cases of condyloma acuminatum, 30 cases of squamous papilloma, 20 cases of hyperkeratosis/acanthosis, 15 cases of epithelial dysplasia, 5 cases of carcinoma in situ, and 15 cases of squamous cell carcinoma. Positive hybridization signals were found in 26 specimens (24.8%). Only HPV-6/11 was detected. HPV DNA occurred significantly more often (p less than 0.005, chi-square analysis) in condyloma acuminatum (100%) and verruca vulgaris (100%) than squamous papilloma (13.3%), hyperkeratotic/acanthotic lesions (10%), and malignant and premalignant lesions (0%). The tongue (19.1%) and labial epithelium (17.1%) were infected most frequently. Nuclear reaction products indicating HPV infection were associated primarily with koilocytes. These results demonstrate the usefulness of commercial biotinylated probes for HPV DNA analysis in routine paraffin-embedded lesion specimens. They confirm HPV involvement in benign lesions of the oral mucosa but fail to associate HPV infection with oral cancer and precancer.

  12. Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

    PubMed Central

    Ren, Fangfang; Zu, Yingbo; Kumar Rajagopalan, Kartik; Wang, Shengnian

    2012-01-01

    Visualizing single DNA dynamics in flow provides a wealth of physical insights in biophysics and complex flow study. However, large signal fluctuations, generated from diversified conformations, deformation history dependent dynamics and flow induced stochastic tumbling, often frustrate its wide adoption in single molecule and polymer flow study. We use a hybrid field microfluidic (HFM) approach, in which an electric field is imposed at desired locations and appropriate moments to balance the flow stress on charged molecules, to effectively regulate the initial conformations and the deformation dynamics of macromolecules in flow. With λ-DNA and a steady laminar shear flow as the model system, we herein studied the performance of HFM on regulating DNA trapping, relaxation, coil-stretch transition, and accumulation. DNA molecules were found to get captured in the focused planes when motions caused by flow, and the electric field were balanced. The trapped macromolecules relaxed in two different routes while eventually became more uniform in size and globule conformations. When removing the electric field, the sudden stretching dynamics of DNA molecules exhibited a more pronounced extension overshoot in their transient response under a true step function of flow stress while similar behaviors to what other pioneering work in steady shear flow. Such regulation strategies could be useful to control the conformations of other important macromolecules (e.g., proteins) and help better reveal their molecular dynamics. PMID:24155864

  13. [Study on molecular hybridization with biotin-labelled HPV 16 DNA probe in human cervical carcinoma].

    PubMed

    Li, Y; Huang, G Q; Mao, T; Huang, Y F; Xiao, H Y; Liu, B L

    1989-09-01

    Biotin-labelled human papillomavirus (HPV) 16 type DNA probe was prepared by the techniques of molecular biology. And dot hybridization technique was used to detect the HPV 16 homologous sequences in the tissues DNA of human cervical carcinoma. The results indicated that 16 cases out of 28 of the human cervical carcinoma tissues were positive. The positive rate was 57%. The other 4 cases of normal uterine cervix tissues were negative. Only 1 in 4 chronic cervicitis tissues showed positive. The HPV 16 plasmid DNA, as the positive control group, showed strong positive, while lambda-phage DNA was negative. The results have shown that the genome of the HPV actually exists in the tissue of the cervical carcinoma and that there is a close relationship between the cervical carcinoma and HPV infection. This experiment adopted the Biotin-labelled HPV 16 DNA probe. And it may provide us with a quick and sensitive method for investigation of the infection of HPV and its role in the carcinogenesis of cervical carcinoma. PMID:2560458

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

    PubMed

    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.

  15. Competitive Metagenomic DNA Hybridization Identifies Host-Specific Microbial Genetic Markers in Cow Fecal Samples†

    PubMed Central

    Shanks, Orin C.; Santo Domingo, Jorge W.; Lamendella, Regina; Kelty, Catherine A.; Graham, James E.

    2006-01-01

    Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities. PMID:16751515

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

    PubMed

    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.

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

    SciTech Connect

    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.

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

    PubMed

    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. PMID:26922047

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

    PubMed

    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

    2016-04-01

    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. PMID:26972953

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

    PubMed

    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

    2016-04-01

    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.

  1. Genetic characterization of inbred lines of Chinese cabbage by DNA markers; towards the application of DNA markers to breeding of F1 hybrid cultivars.

    PubMed

    Kawamura, Kazutaka; Kawanabe, Takahiro; Shimizu, Motoki; Okazaki, Keiichi; Kaji, Makoto; Dennis, Elizabeth S; Osabe, Kenji; Fujimoto, Ryo

    2016-03-01

    Chinese cabbage (Brassica rapa L. var. pekinensis) is an important vegetable in Asia, and most Japanese commercial cultivars of Chinese cabbage use an F1 hybrid seed production system. Self-incompatibility is successfully used for the production of F1 hybrid seeds in B. rapa vegetables to avoid contamination by non-hybrid seeds, and the strength of self-incompatibility is important for harvesting a highly pure F1 seeds. Prediction of agronomically important traits such as disease resistance based on DNA markers is useful. In this dataset, we identified the S haplotypes by DNA markers and evaluated the strength of self-incompatibility in Chinese cabbage inbred lines. The data described the predicted disease resistance to Fusarium yellows or clubroot in 22 Chinese cabbage inbred lines using gene associated or gene linked DNA markers.

  2. Genetic characterization of inbred lines of Chinese cabbage by DNA markers; towards the application of DNA markers to breeding of F1 hybrid cultivars.

    PubMed

    Kawamura, Kazutaka; Kawanabe, Takahiro; Shimizu, Motoki; Okazaki, Keiichi; Kaji, Makoto; Dennis, Elizabeth S; Osabe, Kenji; Fujimoto, Ryo

    2016-03-01

    Chinese cabbage (Brassica rapa L. var. pekinensis) is an important vegetable in Asia, and most Japanese commercial cultivars of Chinese cabbage use an F1 hybrid seed production system. Self-incompatibility is successfully used for the production of F1 hybrid seeds in B. rapa vegetables to avoid contamination by non-hybrid seeds, and the strength of self-incompatibility is important for harvesting a highly pure F1 seeds. Prediction of agronomically important traits such as disease resistance based on DNA markers is useful. In this dataset, we identified the S haplotypes by DNA markers and evaluated the strength of self-incompatibility in Chinese cabbage inbred lines. The data described the predicted disease resistance to Fusarium yellows or clubroot in 22 Chinese cabbage inbred lines using gene associated or gene linked DNA markers. PMID:26862564

  3. Highly sensitive DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization.

    PubMed

    Yu, Xu; Zhang, Zhi-Ling; Zheng, Si-Yang

    2015-04-15

    A novel highly sensitive colorimetric assay for DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization was established. The DNA modified superparamagnetic beads were demonstrated to capture and enrich the target DNA in the hybridization buffer or human plasma. The hybridization chain reaction and enzyme-induced silver metallization on the gold nanoparticles were used as cascade signal amplification for the detection of target DNA. The metalization of silver on the gold nanoparticles induced a significant color change from red to yellow until black depending on the concentration of the target DNA, which could be recognized by naked eyes. This method showed a good specificity for the target DNA detection, with the capabilty to discriminate single-base-pair mismatched DNA mutation (single nucleotide polymorphism). Meanwhile, this approach exhibited an excellent anti-interference capability with the convenience of the magentic seperation and washing, which enabled its usage in complex biological systems such as human blood plasma. As an added benefit, the utilization of hybridization chain reaction and enzyme-induced metallization improved detection sensitivity down to 10pM, which is about 100-fold lower than that of traditional unamplified homogeneous assays.

  4. Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

    PubMed

    Thompson, Brandon L; Birch, Christopher; Li, Jingyi; DuVall, Jacquelyn A; Le Roux, Delphine; Nelson, Daniel A; Tsuei, An-Chi; Mills, Daniel L; Krauss, Shannon T; Root, Brian E; Landers, James P

    2016-08-01

    To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng μL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification. PMID:27250903

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

    PubMed

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

    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 x 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.

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

    PubMed

    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. PMID:27611889

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

    PubMed

    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.

  8. Species-specific identification of Candida krusei by hybridization with the CkF1,2 DNA probe.

    PubMed Central

    Carlotti, A; Couble, A; Domingo, J; Miroy, K; Villard, J

    1996-01-01

    The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amounts as small as 60 to 120 ng of C. krusei target DNA were detected by dot blot hybridization with CkF1,2. It permitted the direct screening of colony blots for early identification. The CkF1,2 probe has potential value as a diagnostic reagent for identifying C. krusei. PMID:8784578

  9. Development of single-cell array for large-scale DNA fluorescence in situ hybridization

    PubMed Central

    Liu, Yingru; Kirkland, Brett; Shirley, James; Wang, Zhibin; Zhang, Peipei; Stembridge, Jacquelyn; Wong, Wilson; Takebayashi, Shin-ichiro; Gilbert, David M.; Lenhert, Steven

    2013-01-01

    DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and easy and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitate analysis of FISH results with the FISH-FINDER computer program. PMID:23370691

  10. Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA-RNA hybrid duplexes: A molecular dynamics simulation study.

    PubMed

    Suresh, Gorle; Priyakumar, U Deva

    2015-09-01

    Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2'-position attribute special properties to nucleic acids when compared to other modifications. 2'-O-methyl modification to deoxyribose sugars of DNA-RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2'-O-methyl group reverses the thermodynamic stability of DNA-RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2'-O-methyl modified counterparts to investigate the effect of 2'-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes.

  11. Reticulate evolution: frequent introgressive hybridization among chinese hares (genus lepus) revealed by analyses of multiple mitochondrial and nuclear DNA loci

    PubMed Central

    2011-01-01

    Background Interspecific hybridization may lead to the introgression of genes and genomes across species barriers and contribute to a reticulate evolutionary pattern and thus taxonomic uncertainties. Since several previous studies have demonstrated that introgressive hybridization has occurred among some species within Lepus, therefore it is possible that introgressive hybridization events also occur among Chinese Lepus species and contribute to the current taxonomic confusion. Results Data from four mtDNA genes, from 116 individuals, and one nuclear gene, from 119 individuals, provides the first evidence of frequent introgression events via historical and recent interspecific hybridizations among six Chinese Lepus species. Remarkably, the mtDNA of L. mandshuricus was completely replaced by mtDNA from L. timidus and L. sinensis. Analysis of the nuclear DNA sequence revealed a high proportion of heterozygous genotypes containing alleles from two divergent clades and that several haplotypes were shared among species, suggesting repeated and recent introgression. Furthermore, results from the present analyses suggest that Chinese hares belong to eight species. Conclusion This study provides a framework for understanding the patterns of speciation and the taxonomy of this clade. The existence of morphological intermediates and atypical mitochondrial gene genealogies resulting from frequent hybridization events likely contribute to the current taxonomic confusion of Chinese hares. The present study also demonstrated that nuclear gene sequence could offer a powerful complementary data set with mtDNA in tracing a complete evolutionary history of recently diverged species. PMID:21794180

  12. A somatic cell hybrid panel for localizing DNA segments near the Huntington's disease gene.

    PubMed

    MacDonald, M E; Anderson, M A; Gilliam, T C; Tranejaerg, L; Carpenter, N J; Magenis, E; Hayden, M R; Healey, S T; Bonner, T I; Gusella, J F

    1987-09-01

    Thirty-four random DNA probes from the terminal half of the human chromosome 4 short arm were further localized within 4pter----p15.1. A panel of somatic cell hybrid lines defining six chromosomal regions within 4pter----p15.1 was constructed using human cell lines containing translocation or deletion chromosomes. The vast majority of the DNA sequences, 32 of 34 or 94%, mapped to the three most proximal regions comprising 4p16.1----4p15.1. Only two probes were localized distal to 4p16.1: one in the region 4p16.3----4p16.1 and one in 4p16.3. D4S10, a polymorphic DNA marker linked to the Huntington's disease defect, has previously been mapped to the terminal region of 4p with conflicting assignments to 4p16.1 and 4p16.3. Analysis of restriction fragment length polymorphisms demonstrated hemizygosity for D4S10 in a patient with Wolf-Hirschhorn syndrome resulting from an unbalanced translocation t(4;8)(p16.3;p23.1), supporting the 4p16.3 localization. Our panel of somatic cell hybrids provides a rapid method for mapping new probes to the same vicinity as that of D4S10. However, the relative paucity of such DNA segments identified here suggests that a more directed approach may be required to generate additional markers near the HD gene.

  13. Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization.

    PubMed

    Reisinger, Jürgen; Rumpler, Silvia; Lion, Thomas; Ambros, Peter F

    2006-04-01

    For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV. PMID:16217752

  14. Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

    PubMed Central

    Tbakhi, A.; Totos, G.; Hauser-Kronberger, C.; Pettay, J.; Baunoch, D.; Hacker, G. W.; Tubbs, R. R.

    1998-01-01

    Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions. Images Figure 1 Figure 2 Figure 3 PMID:9422521

  15. Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays.

    PubMed

    Fixe, F; Branz, H M; Louro, N; Chu, V; Prazeres, D M F; Conde, J P

    2004-07-15

    Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO(2) surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 micros, respectively, about 10(9) times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits. PMID:15142592

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

    PubMed

    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.

  17. Resolution-improved in situ DNA hybridization detection based on microwave photonic interrogation.

    PubMed

    Cao, Yuan; Guo, Tuan; Wang, Xudong; Sun, Dandan; Ran, Yang; Feng, Xinhuan; Guan, Bai-ou

    2015-10-19

    In situ bio-sensing system based on microwave photonics filter (MPF) interrogation method with improved resolution is proposed and experimentally demonstrated. A microfiber Bragg grating (mFBG) is used as sensing probe for DNA hybridization detection. Different from the traditional wavelength monitoring technique, we use the frequency interrogation scheme for resolution-improved bio-sensing detection. Experimental results show that the frequency shift of MPF notch presents a linear response to the surrounding refractive index (SRI) change over the range of 1.33 to 1.38, with a SRI resolution up to 2.6 × 10(-5) RIU, which has been increased for almost two orders of magnitude compared with the traditional fundamental mode monitoring technique (~3.6 × 10(-3) RIU). Due to the high Q value (about 27), the whole process of DNA hybridization can be in situ monitored. The proposed MPF-based bio-sensing system provides a new interrogation method over the frequency domain with improved sensing resolution and rapid interrogation rate for biochemical and environmental measurement.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Automatic on-chip RNA-DNA hybridization assay with integrated phase change microvalves

    NASA Astrophysics Data System (ADS)

    Weng, Xuan; Jiang, Hai; Wang, Junsheng; Chen, Shu; Cao, Honghe; Li, Dongqing

    2012-07-01

    An RNA-DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

  20. Polarization-extinction-based detection of DNA hybridization in situ using a nanoparticle wire-grid polarizer.

    PubMed

    Yu, Hojeong; Oh, Youngjin; Kim, Soowon; Song, Seok Ho; Kim, Donghyun

    2012-09-15

    Metallic wires can discriminate light polarization due to strong absorption of electric fields oscillating in parallel to wires. Here, we explore polarization-based biosensing of DNA hybridization in situ by employing metal target-conjugated nanoparticles to form a wire-grid polarizer (WGP) as complementary DNA strands hybridize. Experimental results using gold nanoparticles of 15 nm diameter to form a WGP of 400 nm period suggest that polarization extinction can detect DNA hybridization with a limit of detection in the range of 1 nM concentration. The sensitivity may be improved by more than an order of magnitude if larger nanoparticles are employed to define WGPs at a period between 400 and 500 nm.

  1. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-09-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.

  2. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

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

    PubMed

    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.

  4. Development of a flow cytometry-based potency assay for measuring the in vitro immunomodulatory properties of mesenchymal stromal cells.

    PubMed

    Ribeiro, Andreia; Ritter, Thomas; Griffin, Matthew; Ceredig, Rhodri

    2016-09-01

    Human bone marrow-derived mesenchymal stromal/stem cells (MSC) have well-documented modulatory effects on multiple immune cell types. Although these effects are linked to their therapeutic benefit in diverse diseases, a reliable, quantitative assay of the immunomodulatory potency of individual human MSC preparations is lacking. The aims of this study were to develop an optimised rapid turnaround, flow cytometry-based whole-blood assay to monitor MSC potency and to validate its application to MSC immunomodulation. A protocol for short-term LPS stimulation of anti-coagulated whole blood samples followed by combined surface CD45/CD14 and intracellular TNF-α staining was initially developed for analysis on a 4 colour desktop cytometer. Optimal monocyte activation was dependent on the presence of extracellular calcium ions thereby precluding the use of EDTA and sodium citrate as anticoagulants. Optimal assay conditions proved to be 1ng/mL ultrapure-LPS added to 10-fold diluted, heparin anti-coagulated whole blood incubated for 6h at 37°C. Under these conditions, addition of human bone marrow-derived MSC (hBM-MSC) from multiple donors resulted in a reproducible, dose-dependent inhibition of LPS-stimulated monocyte TNF-α expression. We conclude that this protocol represents a practical, quantitative assay of a clinically relevant functional effect of hBM-MSCs as well as other immunomodulatory agents. PMID:27451032

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    Xiong, Wanshan; Li, Xiaorong; Fu, Donghui; Mei, Jiaqin; Li, Qinfei; Lu, Guanyuan; Qian, Lunwen; Fu, Yin; Disi, Joseph Onwusemu; Li, Jiana; Qian, Wei

    2013-01-01

    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.

  7. A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes.

    PubMed Central

    Goldkorn, T; Prockop, D J

    1986-01-01

    Single-stranded DNAs (ssDNAs) were covalently bound by a simple and efficient enzymatic method to a solid support matrix and used to develop several new procedures for gene analysis. The novel procedure to prepare a ssDNA stably coupled to a solid support employed T4 DNA ligase to link covalently oligo (dT)-cellulose and (dA)-tailed DNA. Beginning with essentially any double stranded DNA the procedure generates a ssDNA linked by its 5' end to a cellulose matrix in a concentration of over 500 ng per mg. DNA from the plasmid pBR322 (4300 bp) and a fragment of the beta-globin gene (1800 bp) were coupled to the solid support and used for several experiments. The ssDNAs on the cellulose efficiently hybridized with as little as 5 pg of complementary double-stranded DNAs. The DNA hybrids formed on the solid support were specifically and efficiently cleaved by restriction endonucleases. These specific restriction cuts were utilized for the diagnosis of correct sequences. In addition, the ssDNA on the solid support served as an efficient template for the synthesis of complementary ssDNAs. The complementary synthesized ssDNAs were uniformly labeled, more than two kilobases in size, and largely full length. About 85% of the ssDNA linked to cellulose was available for the synthesis of complementary DNA, and after strand-separation, the preparation was reusable for the synthesis of additional complementary DNA. Images PMID:3024131

  8. Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

    PubMed

    Prado, E A; Faivre-Rampant, P; Schneider, C; Darmency, M A

    1996-10-01

    Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.

  9. Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

    SciTech Connect

    Nowotny, Marcin; Cerritelli, Susana M.; Ghirlando, Rodolfo; Gaidamakov, Sergei A.; Crouch, Robert J.; Yang, Wei

    2008-07-09

    Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of {alpha}-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.

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

    PubMed

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

    2013-11-01

    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.

  11. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

  12. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

    Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods.

  13. Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

    PubMed

    Sengiz, Ceren; Congur, Gulsah; Erdem, Arzum

    2015-01-01

    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).

  14. A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers.

    PubMed

    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.

  15. A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

    PubMed Central

    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

  16. Analysis of 5S rDNA organization and variation in polyploid hybrids from crosses of different fish subfamilies.

    PubMed

    Qin, Qinbo; He, Weiguo; Liu, Shaojun; Wang, Jing; Xiao, Jun; Liu, Yun

    2010-07-15

    In this article, sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp (RCC), blunt snout bream (BSB), and their polyploid offspring. Three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) of RCC were characterized by distinct NTS types (designated NTS-I, II, and III for the 83, 220, and 357 bp monomers, respectively). In BSB, only one monomeric 5S rDNA was observed (designated class IV: 188 bp), which was characterized by one NTS type (designated NTS-IV: 68 bp). In the polyploid offspring, the tetraploid (4nRB) hybrids partially inherited 5S rDNA classes from their female parent (RCC); however, they also possessed a unique 5S rDNA sequence (designated class I-L: 203 bp) with a novel NTS sequence (designated NTS-I-L: 83 bp). The characteristic paternal 5S rDNA sequences (class IV) were not observed. The 5S rDNA of triploid (3nRB) hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization. These results first revealed the influence of polyploidy on the organization and evolution of the multigene family of 5S rDNA of fish, and are also useful in clarifying aspects of vertebrate genome evolution.

  17. Hybridization between multiple fence lizard lineages in an ecotone: locally discordant variation in mitochondrial DNA, chromosomes, and morphology.

    PubMed

    Leaché, Adam D; Cole, Charles J

    2007-03-01

    We investigated a hybrid zone between two major lineages of fence lizards (Sceloporus cowlesi and Sceloporus tristichus) in the Sceloporus undulatus species complex in eastern Arizona. This zone occurs in an ecotone between Great Basin Grassland and Conifer Woodland habitats. We analysed spatial variation in mtDNA (N=401; 969 bp), chromosomes (N=217), and morphology (N=312; 11 characters) to characterize the hybrid zone and assess species limits. A fine-scale population level phylogenetic analysis refined the boundaries between these species and indicated that four nonsister mtDNA clades (three belonging to S. tristichus and one to S. cowlesi) are sympatric at the centre of the zone. Estimates of cytonuclear disequilibria in the population closest to the centre of the hybrid zone suggest that the S. tristichus clades are randomly mating, but that the S. cowlesi haplotype has a significant nonrandom association with nuclear alleles. Maximum-likelihood cline-fitting analyses suggest that the karyotype, morphology, and dorsal colour pattern clines are all coincident, but the mtDNA cline is skewed significantly to the south. A temporal comparison of cline centres utilizing karyotype data collected in the early 1970s and in 2002 suggests that the cline may have shifted by approximately 1.5 km to the north over a 30-year period. The recent northward expansion of juniper trees into the Little Colorado River Basin resulting from intense cattle overgrazing provides a plausible mechanism for a shifting hybrid zone and the introgression of the mtDNA haplotypes, which appear to be selectively neutral. It is clear that complex interactions are operating simultaneously in this contact zone, including the formation of hybrids between populations within S. tristichus having diagnostic mtDNA, morphology, karyotypes, and dorsal colour patterns, and secondary contact between these and a distantly related yet morphologically cryptic mtDNA lineage (S. cowlesi). PMID:17305859

  18. Controlled reduction of photobleaching in DNA origami-gold nanoparticle hybrids.

    PubMed

    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.

  19. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    PubMed Central

    Hsu, Joanne H.; Zeng, Hui; Lemke, Kalistyn H.; Polyzos, Aris A.; Weier, Jingly F.; Wang, Mei; Lawin-O’Brien, Anna R.; Weier, Heinz-Ulrich G.; O’Brien, Benjamin

    2013-01-01

    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. PMID:23344021

  20. Not just "a clever way to detect whether DNA really made RNA": The invention of DNA-RNA hybridization and its outcome.

    PubMed

    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. PMID:26209888

  1. Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization

    PubMed Central

    Martinez-Picado, J.; Alsina, M.; Blanch, A. R.; Cerda, M.; Jofre, J.

    1996-01-01

    Methods for specific detection of Vibrio anguillarum in complex microbial communities within diverse marine aquaculture environments were evaluated. A system for the detection of culturable cells based on the combined use of a selective medium and a nonradioactively labeled oligodeoxynucleotide complementary to 16S rRNA was developed. Four hundred fourteen bacterial cultures were evaluated in order to assess the specificity of the method. When both the selective medium and the specific probe gave positive results, the cultures were always identified as V. anguillarum. The selectivity for colony hybridization was 1 V. anguillarum cell in 10,000 total bacterial cells in environmental samples. The utility of the method was also compared with detection by dot blot hybridization of either raw DNA purified from environmental samples or PCR-amplified DNA of 16S rRNA genes, using universal eubacterial primers. The post-PCR hybridization was more sensitive (8 x 10(sup2) cells) than direct hybridization of the whole purified DNA (10(sup6) cells). However, the selective medium-probe combined method was as sensitive as post-PCR hybridization, albeit more specific. PMID:16535233

  2. Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

    PubMed

    Qian, Yong; Wang, Chunyan; Gao, Fenglei

    2015-01-15

    A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis.

  3. Analysis of chromosome band 1p36 alterations by chromosomal in situ suppression hybridization with a microclone DNA bank.

    PubMed

    Zink, D; Weith, A; Martinsson, T; Schwab, M

    1991-09-01

    Alterations of the distal portion of chromosome Ip are a recurrent abnormality of several types of human cancer. In this study we show that chromosomal in situ suppression hybridization with a regional 1p36 DNA bank generated by microdissection and microcloning can be employed to detect translocations involving 1p36.

  4. Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

    PubMed

    Kilianski, Andy; Roth, Pierce A; Liem, Alvin T; Hill, Jessica M; Willis, Kristen L; Rossmaier, Rebecca D; Marinich, Andrew V; Maughan, Michele N; Karavis, Mark A; Kuhn, Jens H; Honko, Anna N; Rosenzweig, C Nicole

    2016-08-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  5. Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.

    PubMed

    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.

  6. [Unidirectional Hybridization of Kaluga Acipenser dauricus Georgi, 1775 and Amur Sturgeon Acipenser schrenckii Brandt, 1869, Inferred from the Mitochondrial DNA Genotyping of Their Natural Hybrids].

    PubMed

    Shedko, S V; Shedko, M B

    2016-03-01

    In 2009 through 2011, among 730 individuals of kaluga and Amur sturgeon collected in the lower reaches of the Amur River and the Amursky Liman, 17 morphologically intermediate individuals (hybrids) with the body length of 56 to 202 cm (median, 81 cm) were identified, including 11 individuals (4.6%) found in 2009, three individuals (1.6%) found in 2010, and three individuals (1.1%), in 2011. In 16 hybrids 819 bp of the mtDNA control regions were sequences and 11 haplotypes were identified. Since all these haplotypes were from the mtDNA lineages of kaluga, it was concluded that hybridization occurred in one direction, kaluga (♀) x Amur sturgeon (♂). This asymmetry could be caused by the large difference in sizes of these species. Since the earlier examined morphologically typical Amur sturgeons showed the absence of alien haplotypes (Shedko, et al., 2015), the absence of the mtDNA introgression is claimed. This can be caused by low viability or sterility of the backcross females (kaluga (♀) x Amur sturgeon (♂)) x Amur sturgeon (♂). The samples of hybrids and typical kaluga individuals demonstrated no differences in the frequency spectra of the mtDNA haplotypes. However, haplotype and nucleotide diversity in the first sample was somewhat higher than in the second one (0.950 versus 0.927 and 0.0054 versus 0.0044, respectively). The data obtained will be useful for population monitoring of kaluga and Amur sturgeon, Amur River endemics, which are classified as critically endangered by the IUCN Red List of Threatened Species. PMID:27281853

  7. [Unidirectional Hybridization of Kaluga Acipenser dauricus Georgi, 1775 and Amur Sturgeon Acipenser schrenckii Brandt, 1869, Inferred from the Mitochondrial DNA Genotyping of Their Natural Hybrids].

    PubMed

    Shedko, S V; Shedko, M B

    2016-03-01

    In 2009 through 2011, among 730 individuals of kaluga and Amur sturgeon collected in the lower reaches of the Amur River and the Amursky Liman, 17 morphologically intermediate individuals (hybrids) with the body length of 56 to 202 cm (median, 81 cm) were identified, including 11 individuals (4.6%) found in 2009, three individuals (1.6%) found in 2010, and three individuals (1.1%), in 2011. In 16 hybrids 819 bp of the mtDNA control regions were sequences and 11 haplotypes were identified. Since all these haplotypes were from the mtDNA lineages of kaluga, it was concluded that hybridization occurred in one direction, kaluga (♀) x Amur sturgeon (♂). This asymmetry could be caused by the large difference in sizes of these species. Since the earlier examined morphologically typical Amur sturgeons showed the absence of alien haplotypes (Shedko, et al., 2015), the absence of the mtDNA introgression is claimed. This can be caused by low viability or sterility of the backcross females (kaluga (♀) x Amur sturgeon (♂)) x Amur sturgeon (♂). The samples of hybrids and typical kaluga individuals demonstrated no differences in the frequency spectra of the mtDNA haplotypes. However, haplotype and nucleotide diversity in the first sample was somewhat higher than in the second one (0.950 versus 0.927 and 0.0054 versus 0.0044, respectively). The data obtained will be useful for population monitoring of kaluga and Amur sturgeon, Amur River endemics, which are classified as critically endangered by the IUCN Red List of Threatened Species.

  8. Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

    PubMed

    Schedl, P; Artavanis-Tsakonas, S; Steward, R; Gehring, W J; Mirault, M E; Goldschmidt-Clermont, M; Moran, L; Tissières, A

    1978-08-01

    The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes. PMID:99246

  9. HyDEn: A Hybrid Steganocryptographic Approach for Data Encryption Using Randomized Error-Correcting DNA Codes

    PubMed Central

    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. PMID:23984392

  10. HyDEn: a hybrid steganocryptographic approach for data encryption using randomized error-correcting DNA codes.

    PubMed

    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.

  11. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

    Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.

  12. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    PubMed

    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.

  13. In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

    PubMed

    Yamaguchi, Tsuyoshi; Kawakami, Shuji; Hatamoto, Masashi; Imachi, Hiroyuki; Takahashi, Masanobu; Araki, Nobuo; Yamaguchi, Takashi; Kubota, Kengo

    2015-07-01

    In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

  14. Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

    PubMed Central

    Zhao, Li; Nakae, Yuki; Qin, Hongmei; Ito, Tadamasa; Kimura, Takahide; Kojima, Hideto; Chan, Lawrence

    2014-01-01

    Summary A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP) has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate) → –N3) in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8) terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL) and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA) through electrostatic attraction, making them promising as nonviral vectors for gene delivery. PMID:24778723

  15. Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

    PubMed Central

    Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L

    2015-01-01

    The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR. PMID:25557623

  16. Human papillomavirus (HPV) DNA in focal epithelial hyperplasia by in situ hybridization.

    PubMed

    Padayachee, A; van Wyk, C W

    1991-05-01

    Eighteen cases of focal epithelial hyperplasia (FEH) were investigated for the presence of human papillomavirus (HPV) group specific antigen by immunocytochemistry and HPV types 1, 6, 11, 13, 16, 18 and 32 by DNA in situ hybridization employing biotinylated probes. Seven (39%) specimens demonstrated the presence of HPV group specific antigen. Fifteen (83%) specimens were positive for HPV DNA: 9 (60%) showed HPV 32, of which 6 were on non-keratinized mucosa and 3 on border of keratinized and non-keratinized mucosa; 5 (33%) showed HPV 13, 4 lesions on keratinized mucosa and 1 on non-keratinized mucosa; 1 (7%) specimen on non-keratinized mucosa showed HPV-11 related. Two specimens on different sites from one patient showed the same HPV type and one patient had, in addition to FEH, a squamous papilloma also demonstrating the same HPV type. Results show a specific HPV distribution pattern in the epithelium indicating areas of high viral concentration adjacent to areas of low or no viral concentration. This study also indicates the possibility of tissue-site specificity or a latent infection and the possibility of a yet unidentified HPV type associated with FEH. It is suggested that future monitoring of patients be carried out with special reference to HPV type and anatomical distribution pattern for FEH lesions.

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

    SciTech Connect

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

    1987-11-01

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

  18. A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

    PubMed

    Dhoble, Abhishek S; Bekal, Sadia; Dolatowski, William; Yanz, Connor; Lambert, Kris N; Bhalerao, Kaustubh D

    2016-11-01

    A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities. PMID:27614579

  19. The detection of Alcelaphine herpesvirus-1 DNA by in situ hybridization of tissues from rabbits affected with malignant catarrhal fever.

    PubMed

    Bridgen, A; Munro, R; Reid, H W

    1992-05-01

    Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host.

  20. DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

    PubMed

    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. PMID:26327272

  1. DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

    PubMed

    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.

  2. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    SciTech Connect

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. )

    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.

  3. DNA hybridization detection with 100 zM sensitivity using piezoelectric plate sensors with an improved noise-reduction algorithm†

    PubMed Central

    Kirimli, Ceyhun E.; Shih, Wei-Heng; Shih, Wan Y.

    2016-01-01

    We have examined real-time, in situ hybridization detection of target DNA (tDNA) in a buffer solution and in urine using 8 μm-thick lead magnesium niobate–lead titanate (PMN–PT) piezoelectric plate sensors (PEPSs) about 1.1–1.2 mm long and 0.45 mm wide with improved 3-mercaptopropyltrimethoxysilane (MPS) insulation and a new multiple-parabola (>50) resonance peak position fitting algorithm. With probe DNA (pDNA) immobilized on the PEPS surface and by monitoring the first width extension mode (WEM) resonance frequency shift we detected tDNA in real time at concentration as low as 1 × 10−19 M in urine (100 zM) with a signal to noise ratio (SNR) of 13 without DNA isolation and amplification at room temperature in 30 min. The present multiple-parabola fitting algorithm increased the detection of SNR by about 10 times compared to those obtained using the raw data and by about 5 times compared to those obtained using single parabola fitting. The detection was validated by in situ follow-up detection and subsequent visualization of fluorescent reporter microspheres (FRMs) coated with reporter DNA complementary to the tDNA but different from the probe pDNA. PMID:24759937

  4. DNA hybridization detection with 100 zM sensitivity using piezoelectric plate sensors with an improved noise-reduction algorithm.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2014-06-01

    We have examined real-time, in situ hybridization detection of target DNA (tDNA) in a buffer solution and in urine using 8 μm-thick lead magnesium niobate-lead titanate (PMN-PT) piezoelectric plate sensors (PEPSs) about 1.1-1.2 mm long and 0.45 mm wide with improved 3-mercaptopropyltrimethoxysilane (MPS) insulation and a new multiple-parabola (>50) resonance peak position fitting algorithm. With probe DNA (pDNA) immobilized on the PEPS surface and by monitoring the first width extension mode (WEM) resonance frequency shift we detected tDNA in real time at concentration as low as 1 × 10(-19) M in urine (100 zM) with a signal to noise ratio (SNR) of 13 without DNA isolation and amplification at room temperature in 30 min. The present multiple-parabola fitting algorithm increased the detection of SNR by about 10 times compared to those obtained using the raw data and by about 5 times compared to those obtained using single parabola fitting. The detection was validated by in situ follow-up detection and subsequent visualization of fluorescent reporter microspheres (FRMs) coated with reporter DNA complementary to the tDNA but different from the probe pDNA. PMID:24759937

  5. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-01-01

    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. PMID:25548939

  6. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    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.

  7. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    PubMed

    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. PMID:26592607

  8. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    PubMed

    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.

  9. M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity

    PubMed Central

    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

  10. Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal.

    PubMed

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

    2016-03-23

    In this work, a novel electrochemical immunoassay protocol has been reported for simultaneous determination of multiple tumor biomarkers based on DNA hybridization chain reaction (HCR) for signal amplification. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were selected as model biomarkers. The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles (Au NPs), secondary antibodies conjugated with DNA concatemer from HCR of primer, auxiliary probe, and signal probe labeled with signal molecules (methyleneblue (MB) and ferrocene (Fc)). In the presence of target biomarkers, the sandwich immunocomplex was formed between the primary antibodies and secondary antibodies bioconjugates carrying numerous signal molecules. As a result, two well-resolved reduction peaks, one was at -0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc; both vs SCE), were obtained in differential pulse voltammetry, and peak currents changed were related to the level of biomarkers. Under optimal conditions, the electrochemical immunoassay exhibited a wide linear response range (0.5 pg mL(-1) to 50 ng mL(-1)) and low detection limits (PSA, 0.17 pg mL(-1); AFP, 0.25 pg mL(-1)) (at S/N = 3). In addition, the immunoassay was evaluated by analyzing simulate human serum sample, and the recoveries obtained were within 99.4-107.6% for PSA and 97.9-108.2% for AFP, indicating the immnuoassay could be applied to the simultaneous detection of AFP and PSA in human serum samples. PMID:26937717

  11. Sorting through the chaff, nDNA gene trees for phylogenetic inference and hybrid identification of annual sunflowers (Helianthus sect. Helianthus).

    PubMed

    Moody, Michael L; Rieseberg, Loren H

    2012-07-01

    The annual sunflowers (Helianthus sect. Helianthus) present a formidable challenge for phylogenetic inference because of ancient hybrid speciation, recent introgression, and suspected issues with deep coalescence. Here we analyze sequence data from 11 nuclear DNA (nDNA) genes for multiple genotypes of species within the section to (1) reconstruct the phylogeny of this group, (2) explore the utility of nDNA gene trees for detecting hybrid speciation and introgression; and (3) test an empirical method of hybrid identification based on the phylogenetic congruence of nDNA gene trees from tightly linked genes. We uncovered considerable topological heterogeneity among gene trees with or without three previously identified hybrid species included in the analyses, as well as a general lack of reciprocal monophyly of species. Nonetheless, partitioned Bayesian analyses provided strong support for the reciprocal monophyly of all species except H. annuus (0.89 PP), the most widespread and abundant annual sunflower. Previous hypotheses of relationships among taxa were generally strongly supported (1.0 PP), except among taxa typically associated with H. annuus, apparently due to the paraphyly of the latter in all gene trees. While the individual nDNA gene trees provided a useful means for detecting recent hybridization, identification of ancient hybridization was problematic for all ancient hybrid species, even when linkage was considered. We discuss biological factors that affect the efficacy of phylogenetic methods for hybrid identification.

  12. 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.

    PubMed

    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.

  13. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    PubMed

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system.

  14. Phylogenetic relationships between annual and perennial species of Helianthus: evolution of a tandem repeated DNA sequence and cytological hybridization experiments.

    PubMed

    Natali, L; Ceccarelli, M; Giordani, T; Sarri, V; Zuccolo, A; Jurman, I; Morgante, M; Cavallini, A; Cionini, P G

    2008-12-01

    The amplification and chromosomal localization of tandem repeated DNA sequences from Helianthus annuus (clone HAG004N15) and the physical organization of ribosomal DNA were studied in annual and perennial species of Helianthus. HAG004N15-related sequences, which did not show amplification in other Asteraceae except for Viguiera multiflora, were redundant in all the Helianthus species tested, but their frequency was significantly higher in perennials than in annuals. These sequences were located at the ends and intercalary regions of all chromosome pairs of annual species. A similar pattern was found in the perennials, but a metacentric pair in their complement was not labelled. Ribosomal cistrons were carried on two chromosome pairs in perennials and on three pairs in annuals except for H. annuus, where rDNA loci were on four pairs. No difference was observed between cultivated H. annuus and its wild accessions in the hybridization pattern of the HAG004N15 and ribosomal probes. These findings support the hypothesis that the separation between annual and perennial Helianthus species occurred through interspecific hybridization involving at least one different parent. However, GISH in H. annuus using genomic DNA from the perennial Helianthus giganteus as blocking DNA failed to reveal different genomic assets in annual and perennial species.

  15. Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

    PubMed

    Albrecht, Bettina; Hausmann, Michael; Zitzelsberger, Horst; Stein, Hubert; Siewert, Jörg Rüdiger; Hopt, Ulrich; Langer, Rupert; Höfler, Heinz; Werner, Martin; Walch, Axel

    2004-07-01

    Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for

  16. Influence of perylenediimide–pyrene supramolecular interactions on the stability of DNA-based hybrids: Importance of electrostatic complementarity

    PubMed Central

    Winiger, Christian B; Langenegger, Simon M; Khorev, Oleg

    2014-01-01

    Summary 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. PMID:25161715

  17. A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

    PubMed Central

    Leach, John C.; Wang, Andrew; Ye, Kaiming; Jin, Sha

    2016-01-01

    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. PMID:26985893

  18. Suppression subtractive hybridization cDNA libraries to identify differentially expressed genes from contrasting fish habitats.

    PubMed

    Straub, Peter F; Higham, Mary L; Tanguy, Arnaud; Landau, Brenda J; Phoel, William C; Hales, L Stanton; Thwing, Theodore K M

    2004-01-01

    Suppression subtractive hybridization complementary DNA libraries identified differentially expressed genes in liver tissue of winter flounder collected from the highly impacted Raritan-Hudson estuary versus those from less industrialized estuaries farther south in New Jersey. Distinct transcript profiles emerged in the fish from these different habitats. A total of 251 clones from the forward (upregulated with anthropogenic impact) and reverse (downregulated with anthropogenic impact) subtracted libraries were sequenced. In the upregulated library immune response transcripts, including complement C-3, C-7, factor H, factor Bf/C2, differentially regulated trout protein 1, and the antimicrobial hepcidin, indicated the pollution-impacted fish were under a high viral or bacterial load. Transcripts for cytochrome P450 1A, P450 3A, and glutathione S-transferase, important components of phase I and II metabolism of xenobiotics, were found in the upregulated-with-pollution library. Vitellogenins I and II and egg envelope protein (zp) appeared to be downregulated. A homologue of the tumor suppressor p33(ING1) (down) and hepatocyte growth factor-like protein (up) may indicate liver damage or hepatocellular carcinoma or hepatoma. These expression patterns, confirmed by quantitative polymerase chain reaction, indicate that transcript analysis is a useful method for assessing the health of local habitats and the organisms therein. PMID:15546050

  19. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  20. Mitochondrial DNA analysis of hybridization between sympatric white-tailed deer and mule deer in west Texas.

    PubMed

    Carr, S M; Ballinger, S W; Derr, J N; Blankenship, L H; Bickham, J W

    1986-12-01

    Sympatric populations of white-tailed deer and mule deer (Odocoileus virginianus and Odocoileus hemionus, respectively) on a west Texas ranch share a common mitochondrial DNA restriction map genotype. Phylogenetic analysis indicates that this genotype is more characteristic of O. virginianus than of O. hemionus. The genotype of west Texas deer differs from that of O. virginianus from South Carolina by five mutational events (1.3% sequence divergence), whereas it differs from that of O. hemionus from California by 17 events (5.5% divergence). We suggest that interspecies hybridization has occurred, primarily between mule deer bucks and white-tailed deer does, with preferential absorption of hybrid offspring into the mule deer gene pool. Introgressive hybridization may be involved in ongoing displacement of mule deer by white-tailed deer in west Texas.

  1. Molecular docking and dynamics simulations on the interaction of cationic porphyrin-anthraquinone hybrids with DNA G-quadruplexes.

    PubMed

    Arba, Muhammad; Kartasasmita, Rahmana E; Tjahjono, Daryono H

    2016-01-01

    A series of cationic porphyrin-anthraquinone hybrids bearing either pyridine, imidazole, or pyrazole rings at the meso-positions have been investigated for their interaction with DNA G-quadruplexes by employing molecular docking and molecular dynamics simulations. Three types of DNA G-quadruplexes were utilized, which comprise parallel, antiparallel, and mixed hybrid topologies. The porphyrin hybrids have a preference to bind with parallel and mixed hybrid structures compared to the antiparallel structure. This preference arises from the end stacking of porphyrin moiety following G-stem and loop binding of anthraquinone tail, which is not found in the antiparallel due to the presence of diagonal and lateral loops that crowd the G-quartet. The binding to the antiparallel, instead, occurred with poorer affinity through both the loop and wide groove. All sites of porphyrin binding were confirmed by 6 ns molecular dynamics simulation, as well as by the negative value of the total binding free energies that were calculated using the MMPBSA method. Free energy analysis shows that the favorable contribution came from the electrostatic term, which supposedly originated from the interaction of either cationic pyridinium, pyrazole, or imidazole groups and the anionic phosphate backbone, and also from the van der Waals energy, which primarily contributed through end stacking interaction.

  2. Silver-enhanced in situ hybridization for detection of polyomavirus DNA in patients with BK virus nephropathy.

    PubMed

    Fritzsche, Florian R; Pianca, Silvio; Gaspert, Ariana; Varga, Zsuzsanna; Wang, Lin; Farrell, Michael P; Chen, Xiao-Bo; Hirsch, Hans H; Springer, Erik; Fehr, Thomas; Myles, Jonathan; Tubbs, Raymond; Moch, Holger

    2011-06-01

    BK virus nephropathy is not an infrequent complication of renal transplantation associated with high rates of graft loss. Although antibodies against SV40 antigen detect different viruses of the polyomavirus family, immunohistochemistry is widely used to confirm the diagnosis of BK virus nephropathy in renal biopsies. Here we aimed to validate the novel silver-enhanced in situ hybridization (SISH) technique for the automated detection of BK virus in renal transplant biopsies. Two different patient cohorts were included. Twenty-nine consecutive patients suspicious for BK virus infection were investigated by SISH and chromogenic in situ hybridization. An additional 26 renal biopsies positive by SV40 immunohistochemistry from 19 patients were analyzed by SISH. Polyomavirus DNA serum levels, as determined by nested PCR analysis, were available for all of these patients. The presence of BK virus DNA in renal tubular cells was identified in 5 of the suspicious cases by both, SISH and chromogenic in situ hybridization . One additional patient was only positive in the SISH. In the second cohort, SISH was positive in all SV40 positive biopsies, but SISH signals were less extensive than SV40 immunohistochemistry. Our results show that the BK virus SISH is an ancillary tool for the detection of polyomavirus DNA in renal biopsies using bright-field microscopy. However, its diagnostic value in comparison with standard immunohistochemistry seems to be limited.

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

    SciTech Connect

    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.

  4. Self-assembly of multiferroic core-shell particulate nanocomposites through DNA-DNA hybridization and magnetic field directed assembly of superstructures

    NASA Astrophysics Data System (ADS)

    Sreenivasulu, Gollapudi; Lochbiler, Thomas A.; Panda, Manashi; Srinivasan, Gopalan; Chavez, Ferman A.

    2016-04-01

    Multiferroic composites of ferromagnetic and ferroelectric phases are of importance for studies on mechanical strain mediated coupling between the magnetic and electric subsystems. This work is on DNA-assisted self-assembly of superstructures of such composites with nanometer periodicity. The synthesis involved oligomeric DNA-functionalized ferroelectric and ferromagnetic nanoparticles, 600 nm BaTiO3 (BTO) and 200 nm NiFe2O4 (NFO), respectively. Mixing BTO and NFO particles, possessing complementary DNA sequences, resulted in the formation of ordered core-shell heteronanocomposites held together by DNA hybridization. The composites were imaged by scanning electron microscopy and scanning microwave microscopy. The presence of heteroassemblies along with core-shell architecture is clearly observed. The reversible nature of the DNA hybridization allows for restructuring the composites into mm-long linear chains and 2D-arrays in the presence of a static magnetic field and ring-like structures in a rotating-magnetic field. Strong magneto-electric (ME) coupling in as-assembled composites is evident from static magnetic field H induced polarization and low-frequency magnetoelectric voltage coefficient measurements. Upon annealing the nanocomposites at high temperatures, evidence for the formation of bulk composites with excellent cross-coupling between the electric and magnetic subsystems is obtained by H-induced polarization and low-frequency ME voltage coefficient. The ME coupling strength in the self-assembled composites is measured to be much stronger than in bulk composites with randomly distributed NFO and BTO prepared by direct mixing and sintering.

  5. Hybridization-sensitive on-off DNA probe: application of the exciton coupling effect to effective fluorescence quenching.

    PubMed

    Ikeda, Shuji; Okamoto, Akimitsu

    2008-06-01

    The design of dyes that emit fluorescence only when they recognize the target molecule, that is, chemistry for the effective quenching of free dyes, must play a significant role in the development of the next generation of functional fluorescent dyes. On the basis of this concept, we designed a doubly fluorescence-labeled nucleoside. Two thiazole orange dyes were covalently linked to a single nucleotide in a DNA probe. An absorption band at approximately 480 nm appeared strongly when the probe was in a single-stranded state, whereas an absorption band at approximately 510 nm became predominant when the probe was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction caused by the formation of an H aggregate between dyes, and as a result, emission from the probe before hybridization was suppressed. Dissociation of aggregates by hybridization with the complementary strand resulted in the disruption of the excitonic interaction and strong emission from the hybrid. This clear change in fluorescence intensity that is dependent on hybridization is useful for visible gene analysis.

  6. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    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.

  7. Specificity of DNA microarray hybridization: characterization, effectors and approaches for data correction

    PubMed Central

    Koltai, Hinanit; Weingarten-Baror, Carmiya

    2008-01-01

    Microarray-hybridization specificity is one of the main effectors of microarray result quality. In the present review, we suggest a definition for specificity that spans four hybridization levels, from the single probe to the microarray platform. For increased hybridization specificity, it is important to quantify the extent of the specificity at each of these levels, and correct the data accordingly. We outline possible effects of low hybridization specificity on the obtained results and list possible effectors of hybridization specificity. In addition, we discuss several studies in which theoretical approaches, empirical means or data filtration were used to identify specificity effectors, and increase the specificity of the hybridization results. However, these various approaches may not yet provide an ultimate solution; rather, further tool development is needed to enhance microarray-hybridization specificity. PMID:18299281

  8. Single-nanoparticle near-infrared surface plasmon resonance microscopy for real-time measurements of DNA hybridization adsorption.

    PubMed

    Halpern, Aaron R; Wood, Jennifer B; Wang, Yong; Corn, Robert M

    2014-01-28

    A novel 814 nm near-infrared surface plasmon resonance (SPR) microscope is used for the real-time detection of the sequence-selective hybridization adsorption of single DNA-functionalized gold nanoparticles. The objective-coupled, high numerical aperture SPR microscope is capable of imaging in situ the adsorption of single polystyrene and gold particles with diameters ranging from 450 to 20 nm onto a 90 μm × 70 μm area of a gold thin film with a time resolution of approximately 1-3 s. Initial real-time SPR imaging (SPRI) measurements were performed to detect the accumulation of 40 nm gold nanoparticles for 10 min onto a gold thin film functionalized with a 100% complementary DNA surface at concentrations from 5 pM to 100 fM by counting individual particle binding events. A 100% noncomplementary DNA surface exhibited virtually no nanoparticle adsorption. In contrast, in a second set of SPRI measurements, two component complementary/noncomplementary mixed DNA monolayers that contained a very small percentage of complementary sequences ranging from 0.1 to 0.001%, showed both permanent and transient hybridization adsorption of the gold nanoparticles that could be tracked both temporally and spatially with the SPR microscope. These experiments demonstrate that SPR imaging measurements of single biofunctionalized nanoparticles can be incorporated into bioaffinity biosensing methods at subpicomolar concentrations.

  9. Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

    PubMed

    Stimpson, D I; Hoijer, J V; Hsieh, W T; Jou, C; Gordon, J; Theriault, T; Gamble, R; Baldeschwieler, J D

    1995-07-01

    The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

  10. Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH).

    PubMed

    Yamaguchi, Tsuyoshi; Fuchs, Bernhard Maximilian; Amann, Rudolf; Kawakami, Shuji; Kubota, Kengo; Hatamoto, Masashi; Yamaguchi, Takashi

    2015-09-01

    Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15 min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.

  11. Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers

    PubMed Central

    2011-01-01

    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 two closely related

  12. AgNP-DNA@GQDs hybrid: new approach for sensitive detection of H2O2 and glucose via simultaneous AgNP etching and DNA cleavage.

    PubMed

    Wang, Lili; Zheng, Jing; Li, Yinhui; Yang, Sheng; Liu, Changhui; Xiao, Yue; Li, Jishan; Cao, Zhong; Yang, Ronghua

    2014-12-16

    A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNP-DNA@GQDs) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (•OH). The formation of AgNP-DNA@GQDs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product •OH between H2O2 and AgNPs could cleave the DNA-bridge and result in the disassembly of AgNP-DNA@GQDs to achieve further signal enhancement. With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6-98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis. PMID:25390796

  13. DNA hybridization evidence for the principal lineages of hummingbirds (Aves:Trochilidae).

    PubMed

    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

  14. DNA hybridization evidence for the principal lineages of hummingbirds (Aves:Trochilidae).

    PubMed

    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

  15. Flow cytometry-based algorithm to analyze the anti-fixed Toxoplasma gondii tachyzoites IgM and IgG reactivity and diagnose human acute toxoplasmosis.

    PubMed

    Silva-dos-Santos, Priscila Pinto; Barros, Geisa Baptista; Mineo, José Roberto; de Oliveira Silva, Deise Aparecida; Menegaz, Mauro Hygino Weinert; Serufo, José Carlos; Dietze, Reynaldo; Martins-Filho, Olindo de Assis; Lemos, Elenice Moreira

    2012-04-30

    In the present study we evaluated the performance of a flow cytometry-based algorithm as a new serological approach to detect antibodies to T. gondii and specific IgG avidity to diagnose acute toxoplasmosis. The results showed that using FC-AFTA-IgM assay, all serum samples from patients with acute toxoplasmosis demonstrated seropositivity, whereas 90% of patients with chronic infection and 100% of non-infected individuals presented negative results. Thus, only 10% of patients with chronic toxoplasmosis showed residual IgM, in contrast with other methodologies used to diagnosis acute toxoplasmosis. On the order hand, FC-AFTA-IgG assay as well as FC-AFTA-IgG subclasses is unlikely to discriminate acute from chronic toxoplasmosis. We have also evaluated the performance of FC-AFTA-IgG avidity as a tool to exclude chronic toxoplasmosis in patients with positive FC-AFTA-IgM. Our data showed an excellent performance of FC-AFTA-IgG avidity employing the cut-off of 60% for Avidity Index (AI) with sensitivity and specificity of 100%. All serum samples from patients presenting acute toxoplasmosis showed low avidity index (AI≤60%), whereas all chronic patients showed high avidity index (AI>60%). The outstanding performance indexes of this novel flow cytometry-based algorithm support its use as a non-conventional alternative serological approach to diagnose human acute toxoplasmosis.

  16. Nuclear corroboration of DNA-DNA hybridization in deep phylogenies of hummingbirds, swifts, and passerines: the phylogenetic utility of ZENK (ii).

    PubMed

    Chubb, Alison L

    2004-01-01

    This paper documents the phylogenetic utility of ZENK at the avian intra-ordinal level using hummingbirds, swifts, and passerines as case studies. ZENK sequences (1.7 kb) were used to reconstruct separate gene trees containing the major lineages of each group, and the three trees were examined for congruence with existing DNA-DNA hybridization trees. The results indicate both that ZENK is an appropriate nuclear marker for resolving relationships deep in the avian tree, and that many relationships within these three particular groups are congruent among the different datasets. Specifically, within hummingbirds there was topological agreement that the major hummingbird lineages diverged in a graded manner from the "hermits," to the "mangoes," to the "coquettes," to the "emeralds," and finally to a sister relationship between the "mountain-gems" and the "bees." Concerning swifts, the deepest divergences were congruent: treeswifts (Hemiprocnidae) were sister to the typical swifts (Apodidae), and the subfamily Apodinae was monophyletic relative to Cypseloidinae. Within Apodinae, however, were short, unresolved branches among the swiftlets, spinetails, and more typical swifts; a finding which coincides with other datasets. Within passerine birds, there was congruent support for monophyly of sub-oscines and oscines, and within sub-oscines, for monophyly of New World groups relative to the Old World lineages. New World sub-oscines split into superfamilies Furnaroidea and Tyrannoidea, with the Tyrannoid relationships completely congruent among ZENK and DNA-DNA hybridization trees. Within Furnaroidea, however, there was some incongruence regarding the positions of Thamnophilidae and Formicariidae. Concerning oscine passerines, both datasets showed a split between Corvida and Passerida and confirmed the traditional membership of passerid superfamilies Muscicapoidea and Passeroidea. Monophyly of Sylvioidea, however, remained uncertain, as did the relationships among the

  17. Nuclear corroboration of DNA-DNA hybridization in deep phylogenies of hummingbirds, swifts, and passerines: the phylogenetic utility of ZENK (ii).

    PubMed

    Chubb, Alison L

    2004-01-01

    This paper documents the phylogenetic utility of ZENK at the avian intra-ordinal level using hummingbirds, swifts, and passerines as case studies. ZENK sequences (1.7 kb) were used to reconstruct separate gene trees containing the major lineages of each group, and the three trees were examined for congruence with existing DNA-DNA hybridization trees. The results indicate both that ZENK is an appropriate nuclear marker for resolving relationships deep in the avian tree, and that many relationships within these three particular groups are congruent among the different datasets. Specifically, within hummingbirds there was topological agreement that the major hummingbird lineages diverged in a graded manner from the "hermits," to the "mangoes," to the "coquettes," to the "emeralds," and finally to a sister relationship between the "mountain-gems" and the "bees." Concerning swifts, the deepest divergences were congruent: treeswifts (Hemiprocnidae) were sister to the typical swifts (Apodidae), and the subfamily Apodinae was monophyletic relative to Cypseloidinae. Within Apodinae, however, were short, unresolved branches among the swiftlets, spinetails, and more typical swifts; a finding which coincides with other datasets. Within passerine birds, there was congruent support for monophyly of sub-oscines and oscines, and within sub-oscines, for monophyly of New World groups relative to the Old World lineages. New World sub-oscines split into superfamilies Furnaroidea and Tyrannoidea, with the Tyrannoid relationships completely congruent among ZENK and DNA-DNA hybridization trees. Within Furnaroidea, however, there was some incongruence regarding the positions of Thamnophilidae and Formicariidae. Concerning oscine passerines, both datasets showed a split between Corvida and Passerida and confirmed the traditional membership of passerid superfamilies Muscicapoidea and Passeroidea. Monophyly of Sylvioidea, however, remained uncertain, as did the relationships among the

  18. A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

    PubMed Central

    Forment, Josep V.; Jackson, Stephen P.

    2016-01-01

    Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

  19. A DNA hybridization system for labeling of neural stem cells with SPIO nanoparticles for MRI monitoring post-transplantation.

    PubMed

    Egawa, Edgar Y; Kitamura, Narufumi; Nakai, Ryusuke; Arima, Yusuke; Iwata, Hiroo

    2015-06-01

    Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 μg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully

  20. Evaluation of the Gibbs Free Energy Changes and Melting Temperatures of DNA/DNA Duplexes Using Hybridization Enthalpy Calculated by Molecular Dynamics Simulation.

    PubMed

    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.

  1. Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes.

    PubMed

    Pioch, Daniel; Jürgen, Britta; Evers, Stefan; Maurer, Karl-Heinz; Hecker, Michael; Schweder, Thomas

    2008-03-01

    Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.

  2. Design of phylum-specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata.

    PubMed

    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.

  3. DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization.

    PubMed

    Horváthová, Eva; Dusinská, Mária; Shaposhnikov, Sergey; Collins, Andrew R

    2004-07-01

    The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a 'matrix-associated region' in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure. PMID:15215325

  4. Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

    PubMed

    Ho, M S; Barr, B C; Tarantal, A F; Lai, L T; Hendrickx, A G; Marsh, A E; Sverlow, K W; Packham, A E; Conrad, P A

    1997-07-01

    Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.

  5. Quantitation of parvovirus B19 DNA sequences by competitive PCR: differential hybridization of the amplicons and immunoenzymatic detection on microplate.

    PubMed

    Gallinella, G; Zerbini, M; Musiani, M; Venturoli, S; Gentilomi, G; Manaresi, E

    1997-04-01

    A competitive PCR assay was developed to quantify B19 DNA sequences. Target and internal standard sequences were co-amplified by the same set of primers. The internal standard competitor was constructed by recombinant PCR and differed from the original genome sequence in a 21-bp mutagenized fragment, internal to the region amplified by the same set of primers. The internal standard competitor was cloned in a plasmid vector and the cloned fragment used in all the experiments. Target and internal standard sequences were labelled with digoxigenin during the co-amplification reaction and the different amplicons were detected in two separate hybridization reactions by biotinylated probes specific for the original 21-bp sequence or the mutagenized one. Hybridized amplicons were captured onto streptavidin-oated microtitre wells and detected by anti-digoxigenin antibodies conjugated to peroxidase. The chromogenic reaction for peroxidase was quantitatively evaluated by optical density determination. The titration curve subsequently developed showed a linear relationship over the range 10(2) to 10(5) genome copies, thus obtaining an exact quantitative evaluation over a wide range together with good sensitivity. Nine reference serum samples positive for B19 DNA and eight negative serum samples were tested by the competitive PCR assay for the quantitation of B19 DNA sequences.

  6. Use of 3,3',5,5' tetramethylbenzidine as new electrochemical indicator of DNA hybridization and its application in genossensor.

    PubMed

    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.

  7. The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing

    PubMed Central

    Chung, Jongsuk; Son, Dae-Soon; Jeon, Hyo-Jeong; Kim, Kyoung-Mee; Park, Gahee; Ryu, Gyu Ha; Park, Woong-Yang; Park, Donghyun

    2016-01-01

    Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications. PMID:27220682

  8. DNA copy number changes in carcinoma in pleomorphic adenoma of the salivary gland: a comparative genomic hybridization study.

    PubMed

    Morio, Takashi; Morimitsu, Yosuke; Hisaoka, Masanori; Makishima, Kazumi; Hashimoto, Hiroshi

    2002-08-01

    Pleomorphic adenoma is the most common benign tumor of the salivary glands and is rarely associated with concurrent epithelial malignancy, which is designated as carcinoma in pleomorphic adenoma (CPA). Genetic abnormalities potentially related to the development of CPA have not been fully investigated. We analyzed DNA copy number changes in each of the adenomatous and carcinomatous components of seven CPA by comparative genomic hybridization using DNA extracted from microdissected tissues of formalin-fixed, paraffin-embedded tumor samples. Carcinomatous components of CPA showed multiple DNA copy number changes at 1-18 different genomic sites (mean 13 sites). Adenomatous components displayed less frequent DNA copy number changes (0-13 sites; mean, 5). In both components, the majority of the changes were gains. The most common recurrent gains in carcinomatous components were seen at 6q (four cases in each), whereas gains at 13q1-2 and 15q1 were most frequently detected in adenomatous components (three cases in each). In five CPA, the same chromosomal regions were involved in the DNA copy number changes detected in both components. Our data suggest that an accumulated or increased number of chromosomal changes including 6q abnormalities may be associated with the development of carcinomatous components in a subset of CPA.

  9. Methods of DNA sequencing by hybridization based on optimizing concentration of matrix-bound oligonucleotide and device for carrying out same

    DOEpatents

    Khrapko, Konstantin R.; Khorlin, Alexandr A.; Ivanov, Igor B.; Ershov, Gennady M.; Lysov, Jury P.; Florentiev, Vladimir L.; Mirzabekov, Andrei D.

    1996-09-03

    A method for sequencing DNA by hybridization that includes the following steps: forming an array of oligonucleotides at such concentrations that either ensure the same dissociation temperature for all fully complementary duplexes or allows hybridization and washing of such duplexes to be conducted at the same temperature; hybridizing said oligonucleotide array with labeled test DNA; washing in duplex dissociation conditions; identifying single-base substitutions in the test DNA by analyzing the distribution of the dissociation temperatures and reconstructing the DNA nucleotide sequence based on the above analysis. A device for carrying out the method comprises a solid substrate and a matrix rigidly bound to the substrate. The matrix contains the oligonucleotide array and consists of a multiplicity of gel portions. Each gel portion contains one oligonucleotide of desired length. The gel portions are separated from one another by interstices and have a thickness not exceeding 30 .mu.m.

  10. Proposition of a Silica Nanoparticle-Enhanced Hybrid Spin-Microcantilever Sensor Using Nonlinear Optics for Detection of DNA in Liquid

    PubMed Central

    Wu, Wen-Hao; Zhu, Ka-Di

    2015-01-01

    We theoretically propose a method based on the combination of a nonlinear optical mass sensor using a hybrid spin-microcantilever and the nanoparticle-enhanced technique, to detect and monitor DNA mutations. The technique theoretically allows the mass of external particles (ssDNA) landing on the surface of a hybrid spin-microcantilever to be detected directly and accurately at 300 K with a mass responsivity 0.137 Hz/ag in situ in liquid. Moreover, combined with the nanoparticle-enhanced technique, even only one base pair mutation in the target DNA sequence can be identified in real time accurately, and the DNA hybridization reactions can be monitored quantitatively. Furthermore, in situ detection in liquid and measurement of the proposed nonlinear optical spin resonance spectra will minimize the experimental errors. PMID:26404276

  11. Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

    SciTech Connect

    Arkesteijn, G.J.A.; Erpelinck, S.L.A.; Martens, A.C.M.; Hagenbeek, A.

    1995-04-01

    Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin- and DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120-fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000. 23 refs., 3 figs., 3 tabs.

  12. Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays.

    PubMed

    Yang, Shengyuan; Liu, Yanmei; Tan, Hui; Wu, Chao; Wu, Zhaoyang; Shen, Guoli; Yu, Ruqin

    2012-03-18

    A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA. PMID:22302154

  13. Extent and degree of hybridization between exotic (Spartina alterniflora) and native (S. foliosa) cordgrass (Poaceae) in California, USA determined by random amplified polymorphic DNA (RAPDs).

    PubMed

    1999-07-01

    Spartina alterniflora, smooth cordgrass, native to the eastern USA, was introduced into south San Francisco Bay approximately 25 years ago. It has spread by purposeful introduction of rooted plants and dispersal of seeds on the tides. Previous work suggested that S. alterniflora was competitively superior to the native California cordgrass, S. foliosa, and that the two species hybridized. The present study determined the spread of S. alterniflora and S. foliosa x alterniflora hybrids in California and examined the degree of hybridization. We used nuclear DNA markers diagnostic for each species to detect the parental species and nine categories of hybrids. The California coast outside San Francisco Bay contained only the native species. All hybrid categories exist in the Bay, implying that several generations of crossing have occurred and that hybridization is bidirectional. Hybrids were found principally near sites of deliberate introduction of the exotic species. Where S. alterniflora was deliberately planted, we found approximately equal numbers of S. alterniflora and hybrid individuals; S. foliosa was virtually absent. Marshes colonized by water-dispersed seed contained the full gamut of phenotypes with intermediate-type hybrids predominating. The proliferation of hybrids could result in local extinction of S. foliosa. What is more, S. alterniflora has the ability to greatly modify the estuary ecosystem to the detriment of other native species and human uses of the Bay. To the extent that they share these engineering abilities, the proliferation of cordgrass hybrids could grossly alter the character of the San Francisco Bay.

  14. Combination of electron microscopic in situ hybridization and anti-DNA antibody labelling reveals a peculiar arrangement of ribosomal DNA in the fibrillar centres of the plant cell nucleolus.

    PubMed

    Yano, Hiroyuki; Sato, Seiichi

    2002-01-01

    The fibrillar centres (FCs) in the nucleoli of Allium cepa usually contained compact dense chromatin, which was always surrounded with light fibrous material (LFM). Distribution of 18S ribosomal DNA (rDNA) in the FCs was examined by in situ hybridization at the light and electron microscopic levels and the results were compared with those obtained by immunogold labelling with anti-DNA antibodies. Anti-DNA antibodies heavily labelled the dense chromatin of the FCs but scarcely labelled the LFM. However, electron microscopic in situ hybridization using the 18S rDNA probe showed that the label in the dense chromatin was extremely weak compared with that obtained by the anti-DNA antibody labelling: the specific label with anti-DNA antibodies of the dense chromatin was about 15 times as much as that of the LFM, whereas the specific label with in situ hybridization in the dense chromatin was only about 1.7 times higher than in the LFM. These results suggest that the rDNA encoding rRNA is preferentially released from the dense chromatin and that non-transcribed intergenic spacers remain in the dense chromatin as the anchoring sites of rDNA. PMID:12227553

  15. Hybridization-based unquenching of DNA hairpins on au surfaces: prototypical "molecular beacon" biosensors.

    PubMed

    Du, Hui; Disney, Matthew D; Miller, Benjamin L; Krauss, Todd D

    2003-04-01

    There is a keen interest in developing techniques for rapid genetic analysis that do not require labeling of an analyte. Here we demonstrate that fluorophore-tagged DNA hairpins attached to gold films can function as immobilized "molecular beacons". Two DNA hairpins incorporating portions of the Staphlococcus aureus FemA and mecR methicillin-resistance genes were attached to a gold substrate. Upon exposure to the complement, a approximately 26-fold increase in fluorescence intensity was measured corresponding to a 96 +/- 5% quenching efficiency. Studies with nonspecific DNA indicate that DNA hairpins immobilized on a gold surface retain their ability to bind complementary DNA sequences selectively.

  16. A Metal-Organic Framework/DNA Hybrid System as a Novel Fluorescent Biosensor for Mercury(II) Ion Detection.

    PubMed

    Wu, Lan-Lan; Wang, Zhuo; Zhao, Shu-Na; Meng, Xing; Song, Xue-Zhi; Feng, Jing; Song, Shu-Yan; Zhang, Hong-Jie

    2016-01-11

    Mercury(II) ions have emerged as a widespread environmental hazard in recent decades. Despite different kinds of detection methods reported to sense Hg(2+) , it still remains a challenging task to develop new sensing molecules to replenish the fluorescence-based apparatus for Hg(2+) detection. This communication demonstrates a novel fluorescent sensor using UiO-66-NH2 and a T-rich FAM-labeled ssDNA as a hybrid system to detect Hg(2+) sensitively and selectively. To the best of our knowledge, it has rarely been reported that a MOF is utilized as the biosensing platform for Hg(2+) assay. PMID:26555340

  17. Identification of DNA homologies among H incompatibility group plasmids by restriction enzyme digestion and Southern transfer hybridization.

    PubMed Central

    Whiteley, M; Taylor, D E

    1983-01-01

    Plasmids belonging to the three HI plasmid incompatibility subgroups were characterized by the use of restriction enzymes and Southern transfer hybridization. A diversity of restriction enzyme patterns was noted among the HI subgroups, and a small amount of DNA homology was observed by probing these digests with a nick-translated HI1 plasmid. Within a single subgroup (HI1 and HI2), similar restriction enzyme patterns were noted. Plasmids of all three HI subgroups and the HII group had a guanine plus cytosine content of 49 to 50 mol%. The IncHII plasmid pHH1508a also showed some homology with the HI1 probe. The DNA homology observed is probably responsible for common phenotypic properties encoded by these plasmids. Images PMID:6314885

  18. Surface-enhanced localized surface plasmon resonance biosensing of avian influenza DNA hybridization using subwavelength metallic nanoarrays

    NASA Astrophysics Data System (ADS)

    Kim, Shin Ae; Byun, Kyung Min; Kim, Kyujung; Jang, Sung Min; Ma, Kyungjae; Oh, Youngjin; Kim, Donghyun; Kim, Sung Guk; Shuler, Michael L.; Kim, Sung June

    2010-09-01

    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.

  19. Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

    PubMed

    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. PMID:27392233

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

    PubMed

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

    2016-01-01

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

  1. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    PubMed

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting.

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

    PubMed

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

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

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

    PubMed Central

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

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

  4. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    PubMed

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting. PMID:27581019

  5. DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae.

    PubMed

    Collado-Romero, Melania; Jiménez-Díaz, Rafael M; Mercado-Blanco, Jesús

    2010-01-01

    The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.

  6. Dual fluorophore PNA FIT-probes--extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA.

    PubMed

    Socher, Elke; Knoll, Andrea; Seitz, Oliver

    2012-09-28

    Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

  7. The antimicrobial lysine-peptoid hybrid LP5 inhibits DNA replication and induces the SOS response in Staphylococcus aureus

    PubMed Central

    2013-01-01

    Background The increase in antibiotic resistant bacteria has led to renewed interest in development of alternative antimicrobial compounds such as antimicrobial peptides (AMPs), either naturally-occurring or synthetically-derived. Knowledge of the mode of action (MOA) of synthetic compounds mimicking the function of AMPs is highly valuable both when developing new types of antimicrobials and when predicting resistance development. Despite many functional studies of AMPs, only a few of the synthetic peptides have been studied in detail. Results We investigated the MOA of the lysine-peptoid hybrid, LP5, which previously has been shown to display antimicrobial activity against Staphylococcus aureus. At concentrations of LP5 above the minimal inhibitory concentration (MIC), the peptoid caused ATP leakage from bacterial cells. However, at concentrations close to the MIC, LP5 inhibited the growth of S. aureus without ATP leakage. Instead, LP5 bound DNA and inhibited macromolecular synthesis. The binding to DNA also led to inhibition of DNA gyrase and topoisomerase IV and caused induction of the SOS response. Conclusions Our data demonstrate that LP5 may have a dual mode of action against S. aureus. At MIC concentrations, LP5 binds DNA and inhibits macromolecular synthesis and growth, whereas at concentrations above the MIC, LP5 targets the bacterial membrane leading to disruption of the membrane. These results add new information about the MOA of a new synthetic AMP and aid in the future design of synthetic peptides with increased therapeutic potential. PMID:23945181

  8. Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

    PubMed Central

    Liu, Fang; Ling, Jian; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    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 was established

  9. Accurate detection of male subclinical genital tract infection via cervical culture and DNA hybridization assay of the female partner.

    PubMed

    Trum, J W; Pannekoek, Y; Spanjaard, L; Bleker, O P; Van Der Veen, F

    2000-02-01

    The accuracy of the PACE2 DNA hybridization assay of the cervix and cervical culture in female partners for the diagnosis of male subclinical genital tract infection were assessed in a male infertility population. A total of 184 men were screened for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis. Seventy-one men were identified with a positive test for one or more of the above mentioned micro-organisms. The overall prevalence of bacterial infection was 39%. Female partners of all men were tested with the PACE2 DNA hybridization assay to detect a C. trachomatis infection. Sensitivity was 100% and specificity was 100%. In 67 female partners (94%) of men who tested positive for U. urealyticum and/or M. hominis, a cervical swab culture was performed. The sensitivity of the cervical swab culture was 100%. In view of the high prevalence of U. urealyticum and M. hominis in the male genital tract and the role these sexually transmitted pathogens may play in infertility, one might question whether all couples should be screened for the presence of these pathogens. Transurethral swab culture after digital prostatic massage is disincentive to men. The cervical culture in their female partner, performed as part of the routine fertility work-up, is a suitable alternative to detect the presence of these micro-organisms in the male genital tract.

  10. Sub-femtomolar electrochemical detection of DNA hybridization based on latex/gold nanoparticle-assisted signal amplification.

    PubMed

    Pinijsuwan, Suttiporn; Rijiravanich, Patsamon; Somasundrum, Mithran; Surareungchai, Werasak

    2008-09-01

    We report a relatively simple electrostatic method for modifying submicrometer-size latex spheres with gold nanoparticles (AuNPs) based on layer-by-layer modification of the latex by polyelectrolytes. The AuNP coverages for 343- and 501-nm-diameter spheres were 4.0 x 10 (10) +/- 1.3 x 10 (10) and 8.2 x 10 (10) +/- 2.7 x 10 (10) particles cm (-2), respectively, which is an increase of 1 order of magnitude on the previously reported coverage at latex-AuNPs using streptavidin-biotin binding (Kawde, A.N.; Wang, J. Electroanalysis 2004, 16, 101-107). Due to the fact that the AuNPs used here are also of a larger size (mean diameter 15.5 +/- 1.6 nm, cf. 5 nm), this represents an increase of 2 orders of magnitude in the number of Au atoms delivered per sphere. The spheres were attached to DNA probes specific to E. coli and used to detect probe hybridization by dissolution of the AuNPs, followed by measurement of Au (3+) ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.5 fM, which is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization.

  11. Aberrant immunity behaviour of hybrid lambda imm21 phages containing the DNA of ColE1-type plasmids.

    PubMed

    Windass, J D; Brammar, W J

    1979-01-01

    Hybrid lambda and lambda imm21 bacteriophages carrying various ColE1-type plasmids have been constructed in vitro. The lambda imm21/plasmid recombinants display aberrant immunity behaviour, giving clear plaques under conditions where the parental phages give turbid ones and being able to grow on homoimmune lysogens. lambda imm lambda/plasmid recombinants show no such unusual behaviour. Studies with hybrids of a lambda imm21 cITS phage carrying pMB9 DNA showed the operation of the plasmid's replication system to be the basic cause of the aberrant immunity behaviour. The plasmid replication system could act as a complete alternative to the phage system during vegetative phage growth. The probable reason that lambda imm21 phages show such altered phenotypes when carrying a functional plasmid replication origin, whereas lambda imm lambda and lambda imm434 (Mukai et al., 1978) phages do not, is the relative ease of titration of the phage 21 repressor to allow transcription from pR21. Various uses are considered for the altered phenotypic behaviour of lambda imm21/ColE1-type plasmid hybrids.

  12. Phenotypic variability confirmed by nuclear ribosomal DNA suggests a possible natural hybrid zone of Triatoma brasiliensis species complex.

    PubMed

    Costa, Jane; Bargues, Maria Dolores; Neiva, Vanessa Lima; Lawrence, Gena G; Gumiel, Marcia; Oliveira, Genova; Cabello, Pedro; Lima, Marli Maria; Dotson, Ellen; Provance, David William; Almeida, Carlos Eduardo; Mateo, Lucia; Mas-Coma, Santiago; Dujardin, Jean Pierre

    2016-01-01

    Triatoma brasiliensis macromelasoma occurs in Pernambuco state, Brazil, which is situated between the distribution areas of Triatoma brasiliensis brasiliensis (north) and Triatoma juazeirensis (south). T. b. macromelasoma displays greater variations in its chromatic phenotype than either T. b. brasiliensis or T. juazeirensis, and patterns reminiscent of one or the other. Experimental crosses from each of these members of the T. brasiliensis species complex generated fertile offspring suggesting that viable hybrids could be present in nature, despite their significant genetic distances. Considering the geographical position of occurrence of the T. b. macromelasoma (in Pernambuco) it was proposed to be an area capable of supporting natural hybridization between T. b. brasiliensis and T. juazeirensis. Since phenotypic variability is expected, this study investigated the existence of intermediate chromatic phenotypes for T. b. macromelasoma in various locations in areas between the T. b. brasiliensis and T. juazeirensis occurrences. Thirteen different color patterns were for the first time characterized and nine of those displayed intermediate phenotypes. Molecular analysis performed using ribosomal DNA intergenic region, grouped all within the T. brasiliensis complex. The intermediate chromatic phenotypes, molecular analysis and experimental crosses all support the distinction of a zone of hybridization that gave rise to the T. b. macromelasoma through homoploidal evolution. PMID:26520796

  13. Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification

    SciTech Connect

    Wimpee, C.F.; Nadeau, T.L.; Nealson, K.H. )

    1991-05-01

    By using two highly conserved regions of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, a cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.

  14. Phenotypic variability confirmed by nuclear ribosomal DNA suggests a possible natural hybrid zone of Triatoma brasiliensis species complex.

    PubMed

    Costa, Jane; Bargues, Maria Dolores; Neiva, Vanessa Lima; Lawrence, Gena G; Gumiel, Marcia; Oliveira, Genova; Cabello, Pedro; Lima, Marli Maria; Dotson, Ellen; Provance, David William; Almeida, Carlos Eduardo; Mateo, Lucia; Mas-Coma, Santiago; Dujardin, Jean Pierre

    2016-01-01

    Triatoma brasiliensis macromelasoma occurs in Pernambuco state, Brazil, which is situated between the distribution areas of Triatoma brasiliensis brasiliensis (north) and Triatoma juazeirensis (south). T. b. macromelasoma displays greater variations in its chromatic phenotype than either T. b. brasiliensis or T. juazeirensis, and patterns reminiscent of one or the other. Experimental crosses from each of these members of the T. brasiliensis species complex generated fertile offspring suggesting that viable hybrids could be present in nature, despite their significant genetic distances. Considering the geographical position of occurrence of the T. b. macromelasoma (in Pernambuco) it was proposed to be an area capable of supporting natural hybridization between T. b. brasiliensis and T. juazeirensis. Since phenotypic variability is expected, this study investigated the existence of intermediate chromatic phenotypes for T. b. macromelasoma in various locations in areas between the T. b. brasiliensis and T. juazeirensis occurrences. Thirteen different color patterns were for the first time characterized and nine of those displayed intermediate phenotypes. Molecular analysis performed using ribosomal DNA intergenic region, grouped all within the T. brasiliensis complex. The intermediate chromatic phenotypes, molecular analysis and experimental crosses all support the distinction of a zone of hybridization that gave rise to the T. b. macromelasoma through homoploidal evolution.

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

    PubMed

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

    2015-10-01

    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.

  16. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21.3) by cDNA hybridization selection.

    PubMed Central

    Goei, V. L.; Parimoo, S.; Capossela, A.; Chu, T. W.; Gruen, J. R.

    1994-01-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. Images Figure 2 Figure 4 PMID:8304341

  17. Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploid formation between Raphanus sativus L. and Brassica alboglabra Bailey

    PubMed Central

    2010-01-01

    Background Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations. Results F1 plants from intergeneric hybridization between Raphanus sativus L. (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) were obtained by hand crosses and subsequent embryo rescue. Random amplification of polymorphic DNA (RAPD) markers were used to identify the F1 hybrid plants. The RAPD data indicated that the hybrids produced specific bands similar to those of parents and new bands that were not present in either parent. Chromosome number variation of somatic cells from allotetraploids in the F4 to F10 generations showed that intensive genetic changes occurred in the early generations of distant hybridization, leading to the formation of mixopolyploids with different chromosome numbers. DNA methylation variation was revealed using MSAP (methylation-sensitive amplification polymorphism), which showed that cytosine methylation patterns changed markedly in the process of hybridization and amphidiploid formation. Differences in cytosine methylation levels demonstrated an epigenetic instability of the allopolyploid of Raphanobrassica between the genetically stable and unstable generations. Conclusions Our results showed that chromosome instability occurred in the early generations of allopolyploidy and then the plants were reverted to largely euploidy in later generations. During this process, DNA methylation changed markedly. These results suggest that, epigenetic mechanisms play an

  18. Optical detection of PNA/DNA hybridization in resonant porous silicon-based devices

    NASA Astrophysics Data System (ADS)

    Rotiroti, Lucia; Arcari, Paolo; Lamberti, Annalisa; Sanges, Carmen; De Tommasi, Edoardo; Rea, Ilaria; Rendina, Ivo; De Stefano, Luca

    2008-04-01

    The development of label-free optical biosensors could have a great impact on life sciences as well as on screening techniques for medical and environmental applications. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone, resulting in an achiral and uncharged mimic. Due to the uncharged nature of PNA, PNA-DNA duplexes show a better thermal stability respect the DNA-DNA equivalents. In this work, we used an optical biosensor, based on the porous silicon (PSi) nanotechnology, to detect PNA-DNA interactions. PSi optical sensors are based on changes of reflectivity spectrum when they are exposed to the target analytes. The porous silicon surface was chemically modified to covalently link the PNA which acts as a very specific probe for its ligand (cDNA).

  19. DNA-Functionalized MFe2O4 (M = Fe, Co, or Mn) Nanoparticles and Their Hybridization to DNA-Functionalized Surfaces

    PubMed Central

    Robinson, David B.; Persson, Henrik H. J.; Zeng, Hao; Li, Guanxiong; Pourmand, Nader; Sun, Shouheng; Wang, Shan X.

    2009-01-01

    Magnetic MFe2O4 (M = Fe, Co, or Mn) nanoparticles with uniform diameters in the 4–20 nm range and with excellent material properties, reported previously, can be rendered soluble in water or aqueous buffers using a combination of alkylphosphonate surfactants and other surfactants such as ethoxylated fatty alcohols or phospholipids. Surfactant-modified oligonucleotides can be incorporated into the particles’ organic shell. The particles can withstand salt concentrations up to 0.3 M, temperatures up to 90 °C, and various operations such as concentration to dryness, column or membrane separations, and electrophoresis. The particles can be selectively hybridized to DNA-functionalized gold surfaces with high coverages using a two-story monolayer structure. These particles may find valuable applications involving the magnetic detection of small numbers of biomolecules using spin valves, magnetic tunnel junctions, or other sensors. PMID:15779990

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

    PubMed Central

    Tønjum, T; Caugant, D A; Bøvre, K

    1992-01-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization. PMID:1452691

  1. Detection of human papillomavirus DNA by in situ hybridization and polymerase chain reaction in human papillomavirus equivocal and dysplastic cervical biopsies.

    PubMed

    Shroyer, K R; Lovelace, G S; Abarca, M L; Fennell, R H; Corkill, M E; Woodard, W D; Davilla, G H

    1993-09-01

    One hundred twenty-one paraffin-embedded cervical biopsy specimens were tested for the presence of human papillomavirus (HPV) DNA by in situ hybridization and polymerase chain reaction. By in situ hybridization using probes for HPV types 6/11, 16/18, 31/33/35, 42/43/44, 51/52, and 45/56, HPV DNA was found in none of 20 normal/squamous metaplasia biopsy specimens, in one of 76 HPV equivocal biopsy specimens, in seven of 12 condyloma/mild dysplasia biopsy specimens, and in 12 of 13 moderate/severe dysplasia biopsy specimens. Polymerase chain reaction using HPV L1 consensus sequence primers followed by filter hybridization of the amplification products was positive for HPV DNA in two of 20 normal/squamous metaplasia biopsy specimens, in 23 of 76 HPV equivocal biopsy specimens, in eight of 12 condyloma/mild dysplasia biopsy specimens, and in 12 of 13 moderate/severe dysplasia biopsy specimens. Among biopsies that tested positive by polymerase chain reaction but that were negative by in situ hybridization, the most commonly identified HPV was type 16. We conclude that although HPV equivocal biopsy specimens contain HPV DNA more frequently than histologically normal tissue, the majority of biopsy specimens in this category test negative for HPV DNA. The clinical significance of a positive test for HPV, in the absence of unequivocal histologic changes, remains to be determined.

  2. Comparison of hybridization behavior between double and single strands of targets and the application of asymmetric PCR targets in cDNA microarray.

    PubMed

    Wei, Qing; Liu, Sanzhen; Huang, Jianfeng; Mao, Xueying; Chu, Xiaohui; Wang, Yu; Qiu, MinYan; Mao, Yumin; Xie, Yi; Li, Yao

    2004-07-31

    Double stranded targets on the cDNA microarray contain representatives of both the coding and noncoding strands, which will introduce hybridization competition with probes. Here, the effect of double and single strands of targets on the signal intensity and the ratios of Cy5/Cy3 within the same slide were compared. The results show that single stranded targets can increase the hybridization efficiency without changing the Cy5/Cy3 ratio. Based on these results, a new strategy was established by generating cDNA targets with asymmetric PCR, instead of conventional PCR, to increase the sensitivity of the cDNA microarray. Furthermore, the feasibility of this approach was validated. The results indicate that the cDNA microarray system based on asymmetric PCR is more sensitive, with no decrease in the reliability and reproducibility as compared with that based on conventional symmetric PCR.

  3. Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement.

    PubMed

    Bai, Yu-Hui; Li, Jin-Yi; Xu, Jing-Juan; Chen, Hong-Yuan

    2010-07-01

    A novel method is described for the highly effective amplifying electrochemical response of DNA based on oligonucleotides functionalized with Au/Fe(3)O(4) nanocomposites by the aid of silver (Ag) enhancement. Via electrostatic layer-by-layer (LBL) assembly, the prepared Fe(3)O(4) nanoparticles form nano-clusters coated with a bilayer composed of polystyrene sulfonate sodium salt (PSS) and poly(diallyldimethylammonium chloride) (PDDA), which are in favor of adsorbing lots of gold nanoparticles (AuNPs) on the surface. The application of magnetic Fe(3)O(4) made the procedures much more simple, convenient and feasible. The resulting composites were then used as labels via the Au-S bond for the DNA hybridization, followed by catalytic deposition of silver on the gold tags. Such an assay is then combined with a sensitive anodic stripping voltammetry (ASV) measurement of multiple silver nanoparticle tracers. A 27-mer sequence DNA target is detected at a glassy carbon (GC) electrode with a detection limit down to ca. 100 aM, which is 800 times lower than that obtained using gold nanoparticles only as labels in the control experiments. This Fe(3)O(4)/PSS/PDDA/Au composite offers a great promising future for the ultrasensitive detection of other biorecognition events.

  4. DanQ: a hybrid convolutional and recurrent deep neural network for quantifying the function of DNA sequences.

    PubMed

    Quang, Daniel; Xie, Xiaohui

    2016-06-20

    Modeling the properties and functions of DNA sequences is an important, but challenging task in the broad field of genomics. This task is particularly difficult for non-coding DNA, the vast majority of which is still poorly understood in terms of function. A powerful predictive model for the function of non-coding DNA can have enormous benefit for both basic science and translational research because over 98% of the human genome is non-coding and 93% of disease-associated variants lie in these regions. To address this need, we propose DanQ, a novel hybrid convolutional and bi-directional long short-term memory recurrent neural network framework for predicting non-coding function de novo from sequence. In the DanQ model, the convolution layer captures regulatory motifs, while the recurrent layer captures long-term dependencies between the motifs in order to learn a regulatory 'grammar' to improve predictions. DanQ improves considerably upon other models across several metrics. For some regulatory markers, DanQ can achieve over a 50% relative improvement in the area under the precision-recall curve metric compared to related models. We have made the source code available at the github repository http://github.com/uci-cbcl/DanQ.

  5. Nanosilver-based surface-enhanced Raman spectroscopic determination of DNA methyltransferase activity through real-time hybridization chain reaction.

    PubMed

    Hu, Ping Ping; Liu, Hui; Zhen, Shu Jun; Li, Chun Mei; Huang, Cheng Zhi

    2015-11-15

    In this manuscript, a nanosilver enhanced SERS strategy was successfully constructed for the determination of DNA methyltransferase activity in soulution combined with hybridization chain reaction (HCR). The proposed method was mainly on the basis of excellent separation ability of magnetic microparticles (MMPs), HCR as signal amplification unit and assembled AgNPs as enhancement substrate. In the presence of M. SssI MTase, the duplex sequence (5'-CCGG-3') tethered to MMPs was methylated, which cannot be cleaved by HpaII endonuclease. The resulted DNA skeleton captured on MMPs then triggered the HCR reaction, generated a polymerized and extended symmetrical sequence, in which more biotin terminal was available for the conjugation of AgNPs-SA, leading to significantly amplified SERS response. When it was used to analyze M. SssI activity, a linear equation ∆ISERS=1215.32+446.80 cM.SssI was obtained with the M. SssI activity ranged from 0.1 to 10.0 U with the correlation coefficient (r(2)) of 0.97. The most important advantage of this method is the combination of SERS and HCR in solution for the first time and its good selectivity, which enabled the detection of even one-base mismatched sequence. The new assay method holds great promising application to be a versatile platform for sensitive, high-throughput detection, and the screening of new anticancer drugs on DNA MTase. PMID:26086442

  6. DanQ: a hybrid convolutional and recurrent deep neural network for quantifying the function of DNA sequences.

    PubMed

    Quang, Daniel; Xie, Xiaohui

    2016-06-20

    Modeling the properties and functions of DNA sequences is an important, but challenging task in the broad field of genomics. This task is particularly difficult for non-coding DNA, the vast majority of which is still poorly understood in terms of function. A powerful predictive model for the function of non-coding DNA can have enormous benefit for both basic science and translational research because over 98% of the human genome is non-coding and 93% of disease-associated variants lie in these regions. To address this need, we propose DanQ, a novel hybrid convolutional and bi-directional long short-term memory recurrent neural network framework for predicting non-coding function de novo from sequence. In the DanQ model, the convolution layer captures regulatory motifs, while the recurrent layer captures long-term dependencies between the motifs in order to learn a regulatory 'grammar' to improve predictions. DanQ improves considerably upon other models across several metrics. For some regulatory markers, DanQ can achieve over a 50% relative improvement in the area under the precision-recall curve metric compared to related models. We have made the source code available at the github repository http://github.com/uci-cbcl/DanQ. PMID:27084946

  7. Flexible microfabricated film sensors for the in situ quantum dot-based voltammetric detection of DNA hybridization in microwells.

    PubMed

    Kokkinos, Christos; Economou, Anastasios; Speliotis, Thanasis; Petrou, Panagiota; Kakabakos, Sotirios

    2015-01-20

    A new flexible miniaturized integrated device was microfabricated for the in situ ultrasensitive voltammetric determination of DNA mutation in a microwell format, using quantum dots (QDs) labels. The integrated device consisted of thin Bi, Ag, and Pt films (serving as the working, reference, and counter electrode, respectively) deposited by sputtering on a flexible polyimide substrate. A DNA assay was employed in microwell format, where an immobilized complementary oligonucleotide probe hybridized with the biotinylated target oligonucleotide followed by reaction with streptavidin-conjugated PbS QDs. After the acidic dissolution of the QDs, the flexible sensor was rolled and inserted into the microwell and the Pb(II) released was determined in situ by anodic stripping voltammetry. Since the analysis took place directly in the microwell, the volume of the working solution was only 100 μL and the target DNA could be detected at a concentration down to 1.1 fmol L(-1). The proposed flexible microdevice addresses the restrictions of conventional rigid electrodes while it provides a low cost integrated transducer for the ultrasensitive detection of important biomolecules. PMID:25514352

  8. Hypoxia-induced regulation of MAPK phosphatase-1 as identified by subtractive suppression hybridization and cDNA microarray analysis.

    PubMed

    Seta, K A; Kim, R; Kim, H W; Millhorn, D E; Beitner-Johnson, D

    2001-11-30

    Subtractive suppression hybridization was used to generate a cDNA library enriched in cDNA sequences corresponding to mRNA species that are specifically up-regulated by hypoxia (6 h, 1% O(2)) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPK phosphatase-1 (MKP-1) was highly represented in this library. Clones were arrayed on glass slides to create a hypoxia-specific cDNA microarray chip. Microarray, northern blot, and western blot analyses confirmed that MKP-1 mRNA and protein levels were up-regulated by hypoxia by approximately 8-fold. The magnitude of the effect of hypoxia on MKP-1 was approximately equal to that induced by KCl depolarization and much larger than the effects of either epidermal growth factor or nerve growth factor on MKP-1 mRNA levels. In contrast to the calcium-dependent induction of MKP-1 by KCl depolarization, the effect of hypoxia on MKP-1 persisted under calcium-free conditions. Cobalt and deferoxamine also increased MKP-1 mRNA levels, suggesting that hypoxia-inducible factor proteins may play a role in the regulation of MKP-1 by hypoxia. Pretreatment of cells with SB203580, which inhibits p38 kinase activity, significantly reduced the hypoxia-induced increase in MKP-1 RNA levels. Thus, hypoxia robustly increases MKP-1 levels, at least in part through a p38 kinase-mediated mechanism. PMID:11577072

  9. Density functional study of the phosphate diester hydrolysis of RNA in RNA/DNA hybrid by RNase HI

    NASA Astrophysics Data System (ADS)

    Kita, Makoto; Nakamura, Haruki; Takano, Yu

    2014-02-01

    Ribonuclease HI (RNase HI) catalyses the non-specific hydrolysis of RNA in an RNA/DNA hybrid. This enzyme is found in almost all organisms and involved in replication initiation and DNA topology restoration. A similar fold has been observed in other enzymes such as DNA transposases. In particular, RNases HI has emerged as important therapeutic targets because the enzymatic activity is absolutely required for proliferation of HIV and other retroviruses. The X-ray crystallographic structures of RNase HI revealed that the Mg2+ ion is essential for the enzymatic reaction and that Asp and Glu coordinate to the Mg2+ ion. There are, however, controversies about the catalytic mechanism of RNase HI, the number of Mg2+ ions, the proton transfer pathway, and the protonation states of the active site residues. In the present study, we have explored the hydrolysis of the phosphate diester group of RNA by RNase HI using density functional theory to elucidate how many Mg2+ ions are required for the catalysis of RNase HI. Our computation demonstrates that both one- and two-metal models show the stepwise hydrolysis pathway via a pentacovalent intermediate. However, the activation barrier of the two-metal model is lower than that of the one-metal model by 11.7 kcal/mol.

  10. Cyclometalated iridium complex-based label-free photoelectrochemical biosensor for DNA detection by hybridization chain reaction amplification.

    PubMed

    Li, Chunxiang; Wang, Hongyang; Shen, Jing; Tang, Bo

    2015-04-21

    Photoactive material is the most crucial factor which intimately determines analytical performances of the photoelectrochemical sensor. On the basis of the high affinity of dipyrido [3,2-a:2',3'-c] phenazine (dppz) with DNA helix, a novel photoactive intercalator, [(ppy)2Ir(dppz)](+)PF6(-)(ppy = 2-phenylpyridine and dppz = dipyrido [3,2-a:2',3'-c] phenazine) was prepared and characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, and cyclic voltammetry. The photoelectrochemical properties of the as-prepared iridium(III) complex immobilized on the ITO electrode was investigated. Either cathodic or anodic photocurrent generation can be observed when triethanolamine (TEOA) or dissolved O2 is used as a sacrificial electron donor/acceptor, respectively. The probable photocurrent-generation mechanisms are speculated. A highly sensitive iridium(III) complex-based photoelectrochemical sensor was proposed for DNA detection via hybridization chain reaction (HCR) signal amplification. Under optimal conditions, the biosensor was found to be linearly proportional to the logarithm of target DNA concentration in the range from 0.025 to 100 pmol L(-1) with a detection limit of 9.0 fmol L(-1) (3σ). Moreover, the proposed sensor displayed high selectivity and good reproducibility, demonstrating efficient and stable photoelectric conversion ability of the Ir(III) complex. PMID:25816127

  11. DanQ: a hybrid convolutional and recurrent deep neural network for quantifying the function of DNA sequences

    PubMed Central

    Quang, Daniel; Xie, Xiaohui

    2016-01-01

    Modeling the properties and functions of DNA sequences is an important, but challenging task in the broad field of genomics. This task is particularly difficult for non-coding DNA, the vast majority of which is still poorly understood in terms of function. A powerful predictive model for the function of non-coding DNA can have enormous benefit for both basic science and translational research because over 98% of the human genome is non-coding and 93% of disease-associated variants lie in these regions. To address this need, we propose DanQ, a novel hybrid convolutional and bi-directional long short-term memory recurrent neural network framework for predicting non-coding function de novo from sequence. In the DanQ model, the convolution layer captures regulatory motifs, while the recurrent layer captures long-term dependencies between the motifs in order to learn a regulatory ‘grammar’ to improve predictions. DanQ improves considerably upon other models across several metrics. For some regulatory markers, DanQ can achieve over a 50% relative improvement in the area under the precision-recall curve metric compared to related models. We have made the source code available at the github repository http://github.com/uci-cbcl/DanQ. PMID:27084946

  12. Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

    PubMed

    Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

    2012-03-01

    Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

  13. A strategy for antimicrobial regulation based on fluorescent conjugated oligomer-DNA hybrid hydrogels.

    PubMed

    Cao, Ali; Tang, Yanli; Liu, Yue; Yuan, Huanxiang; Liu, Libing

    2013-06-21

    New fluorescent oligo(phenylene ethynylene)-DNA hydrogels have been prepared and used for the controllable biocidal activity driven by DNase. This study opens a new way of controllable drug release and antimicrobial regulation.

  14. COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC GENETIC MARKERS IN HUMAN FECAL MICROBIAL COMMUNITIES

    EPA Science Inventory

    Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, the use of these approaches to discern key genomic differences between natural microbial communities remains prohibitively expensive for mo...

  15. A rapid procedure to detect in situ DNA/RNA hybrids

    SciTech Connect

    Reddy, A.R.; Sofer, W.

    1981-12-15

    A new method was developed for detecting DNA/RNA hydrids formed in situ using anti-DNA/RNA antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesized in vitro from cloned Drosophila histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.

  16. Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

    PubMed Central

    Barakat, Tahsin Stefan; Gribnau, Joost

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

  17. Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

    PubMed Central

    Ranjan, Rajiv; Patro, Sunita; Pradhan, Bhubaneswar; Kumar, Alok; Maiti, Indu B.; Dey, Nrisingha

    2012-01-01

    Background Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. Methodology Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. Significance/Conclusion Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the

  18. DNA-Hybrid-Gated Photothermal Mesoporous Silica Nanoparticles for NIR-Responsive and Aptamer-Targeted Drug Delivery.

    PubMed

    Zhang, Yuanxin; Hou, Zhiyao; Ge, Yakun; Deng, Kerong; Liu, Bei; Li, Xuejiao; Li, Quanshun; Cheng, Ziyong; Ma, Ping'an; Li, Chunxia; Lin, Jun

    2015-09-23

    Near-infrared light is an attractive stimulus due to its noninvasive and deep tissue penetration. Particularly, NIR light is utilized for cancer thermotherapy and on-demand release of drugs by the disruption of the delivery carriers. Here we have prepared a novel NIR-responsive DNA-hybrid-gated nanocarrier based on mesoporous silica-coated Cu1.8S nanoparticles. Cu1.8S nanoparticles, possessing high photothermal conversion efficiency under a 980 nm laser, were chosen as photothermal agents. The mesoporous silica structure could be used for drug storage/delivery and modified with aptamer-modified GC-rich DNA-helix as gatekeepers, drug vectors, and targeting ligand. Simultaneously, the as-produced photothermal effect caused denaturation of DNA double strands, which triggered the drug release of the DNA-helix-loaded hydrophilic drug doxorubicin and mesopore-loaded hydrophobic drug curcumin, resulting in a synergistic therapeutic effect. The Cu1.8S@mSiO2 nanocomposites endocytosed by cancer cells through the aptamer-mediated mode are able to generate rational release of doxorubicin/curcumin under NIR irradiation, strongly enhancing the synergistic growth-inhibitory effect of curcumin against doxorubicin in MCF-7 cells, which is associated with a strong mitochondrial-mediated cell apoptosis progression. The underlying mechanism of apoptosis showed a strong synergistic inhibitory effect both on the expression of Bcl-2, Bcl-xL, Mcl-1, and upregulated caspase 3/9 activity and on the expression level of Bak and Bax. Therefore, Cu1.8S@mSiO2 with efficient synergistic therapeutic efficiency is a potential multifunctional cancer therapy nanoplatform. PMID:26325285

  19. Implications of Hybridization, NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

    PubMed Central

    Ermakov, Oleg A.; Simonov, Evgeniy; Surin, Vadim L.; Titov, Sergey V.; Brandler, Oleg V.; Ivanova, Natalia V.; Borisenko, Alex V.

    2015-01-01

    The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’. PMID:25617768

  20. DNA sequence-dependent morphological evolution of silver nanoparticles and their optical and hybridization properties.

    PubMed

    Wu, Jiangjiexing; Tan, Li Huey; Hwang, Kevin; Xing, Hang; Wu, Peiwen; Li, Wei; Lu, Yi

    2014-10-29

    A systematic investigation of the effects of different DNA sequences on the morphologies of silver nanoparticles (AgNPs) grown from Ag nanocube seeds is reported. The presence of 10-mer oligo-A, -T, and -C directed AgNPs growth from cubic seeds into edge-truncated octahedra of different truncation extents and truncated tetrahedral AgNPs, while AgNPs in the presence of oligo-G remained cubic. The shape and morphological evolution of the nanoparticle growth for each system is investigated using SEM and TEM and correlated with UV-vis absorption kinetic studies. In addition, the roles of oligo-C and oligo-G secondary structures in modulating the morphologies of AgNPs are elucidated, and the morphological evolution for each condition of AgNPs growth is proposed. The shapes were found to be highly dependent on the binding affinity of each of the bases and the DNA secondary structures, favoring the stabilization of the Ag{111} facet. The AgNPs synthesized through this method have morphologies and optical properties that can be varied by using different DNA sequences, while the DNA molecules on these AgNPs are also stable against glutathione. The AgNP functionalization can be realized in a one-step synthesis while retaining the biorecognition ability of the DNA, which allows for programmable assembly. PMID:25243485

  1. A reactive peptidic linker for self-assembling hybrid quantum dot-DNA bioconjugates.

    PubMed

    Medintz, Igor L; Berti, Lorenzo; Pons, Thomas; Grimes, Amy F; English, Douglas S; Alessandrini, Andrea; Facci, Paolo; Mattoussi, Hedi

    2007-06-01

    Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.

  2. Implications of hybridization, NUMTs, and overlooked diversity for DNA Barcoding of Eurasian ground squirrels.

    PubMed

    Ermakov, Oleg A; Simonov, Evgeniy; Surin, Vadim L; Titov, Sergey V; Brandler, Oleg V; Ivanova, Natalia V; Borisenko, Alex V

    2015-01-01

    The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5-4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic 'mini-barcodes'.

  3. Real time detection of DNA.RNA hybridization in living cells.

    PubMed

    Sokol, D L; Zhang, X; Lu, P; Gewirtz, A M

    1998-09-29

    Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be >/=1 x 10(-1) ag of MB, or approximately 10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that real-time visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.

  4. New high performance hybrid magnet plates for DNA separation andbio-technology applications

    SciTech Connect

    Humphries, David; Pollard, Martin; Elkin, Chris; Petermann, Karl; Reiter, Charles; Cepeda, Mario

    2004-08-02

    A new class of magnet plates for biological and industrial applications has recently been developed at the D.O.E. Joint Genome Institute and Lawrence Berkeley National Laboratory (JGI/LBNL). These devices utilize hybrid technology that combines linear permanent magnet material and ferromagnetic material to produce significantly higher fields and gradients than currently available commercial magnet plates. These hybrid structures incorporate ferromagnetic poles that can be easily shaped to produce complex field distributions for specialized applications. The higher maximum fields and strong gradients of the hybrid structures result in greater holding forces on magnetized targets that are being processed as well as faster draw-down. Current development versions of these magnet plates have exhibited maximum fields in excess of 9000.0 Gauss. The design of these structures is easily scalable to allow for field increases to significantly above 1.0 tesla (10000.0gauss). Author's note: 11000.0 Gauss peak fields have been achieved as of January 2005.

  5. Coupled equilibrium model of hybridization error for the DNA microarray and tag-antitag systems.

    PubMed

    Rose, John A; Deaton, Russell J; Hagiya, Masami; Suyama, Akira

    2007-03-01

    In this work, a detailed coupled equilibrium model is presented for predicting the ensemble average probability of hybridization error per chip-hybridized input strand, providing the first ensemble average method for estimating postannealing microarray/TAT system error rates. Following a detailed presentation of the model and implementation via the software package NucleicPark, under a mismatched statistical zipper model of duplex formation, error response is simulated for both mean-energy and randomly encoded TAT systems versus temperature and input concentration. Limiting expressions and simulated model behavior indicate the occurrence of a transition in hybridization error response, from a logarithmically convex function of temperature for excess inputs (high-error behavior), to a monotonic, log-linear function of temperature for dilute inputs (low-error behavior), a novel result unpredicted by uncoupled equilibrium models. Model scaling behavior for random encodings is investigated versus system size and strand-length. Application of the model to TAT system design is also undertaken, via the in silico evolution of a high-fidelity 100-strand TAT system, with an error response improved by nine standard deviations over the performance of the mean random encoding. PMID:17393846

  6. Coupled equilibrium model of hybridization error for the DNA microarray and tag-antitag systems.

    PubMed

    Rose, John A; Deaton, Russell J; Hagiya, Masami; Suyama, Akira

    2007-03-01

    In this work, a detailed coupled equilibrium model is presented for predicting the ensemble average probability of hybridization error per chip-hybridized input strand, providing the first ensemble average method for estimating postannealing microarray/TAT system error rates. Following a detailed presentation of the model and implementation via the software package NucleicPark, under a mismatched statistical zipper model of duplex formation, error response is simulated for both mean-energy and randomly encoded TAT systems versus temperature and input concentration. Limiting expressions and simulated model behavior indicate the occurrence of a transition in hybridization error response, from a logarithmically convex function of temperature for excess inputs (high-error behavior), to a monotonic, log-linear function of temperature for dilute inputs (low-error behavior), a novel result unpredicted by uncoupled equilibrium models. Model scaling behavior for random encodings is investigated versus system size and strand-length. Application of the model to TAT system design is also undertaken, via the in silico evolution of a high-fidelity 100-strand TAT system, with an error response improved by nine standard deviations over the performance of the mean random encoding.

  7. Label free detection of DNA on Au/ZnO/Ag hybrid structure based SERS substrate

    NASA Astrophysics Data System (ADS)

    Pal, Anil Kumar; Mohan, D. Bharathi

    2016-04-01

    Au/ZnO/Ag based SERS substrate was fabricated for the label free detection of DNA of Escherichia Coli bacteria. The SERS substrate was fabricated by growing ZnO nanorod arrays on thermally evaporated ultrathin Ag film of 5 nm thickness using hydrothermal process. Non-spherical like Au nanoparticles were decorated on ZnO nanorod arrays by sputtering technique with sputtering time of 45 sec. The surface of Au/ZnO/Ag was observed to be nearly superhydrophobic exhibiting the contact angle of 144 °. A low volume (5 µl) of aqueous solution of DNA of laboratory strain Escherichia Coli with very low concentration was adsorbed on fabricated SERS substrate by drop casting. The SERS detection of DNA molecules was achieved up to lower concentration of 10-8 M due to strong local electric field enhancement at the nanometer gap among Au nanoparticles and superhydrophobic nature of Au/ZnO/Ag surface.

  8. Solid-phase synthesis and hybridization properties of DNA containing sulfide-linked dinucleosides.

    PubMed Central

    Kawai, S H; Wang, D; Giannaris, P A; Damha, M J; Just, G

    1993-01-01

    Oligodeoxyribonucleotides incorporating non-hydrolyzable dialkyl sulfide linked thymidine dimers (TsT) were synthesized chemically by the solid-phase approach. The sulfide dimer TsT was stable to degradation by snake-venom phosphodiesterase, calf spleen phosphodiesterase, Nuclease P1 and Nuclease S1. Thermal denaturation analysis indicated that the incorporation of TsT dimers into DNA weakened, but did not prevent, binding to complementary DNA and RNA over a wide range of salt concentrations (10 mM to 2 M NaCl). Images PMID:8464740

  9. Isothermal DNA origami folding: avoiding denaturing conditions for one-pot, hybrid-component annealing

    NASA Astrophysics Data System (ADS)

    Kopielski, Andreas; Schneider, Anne; Csáki, Andrea; Fritzsche, Wolfgang

    2015-01-01

    The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously.The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly

  10. Comparison between the complete mtDNA sequences of the blue and the fin whale, two species that can hybridize in nature.

    PubMed

    Arnason, U; Gullberg, A

    1993-10-01

    The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 bp long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0-7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of > or = 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be.

  11. A label-free colorimetric platform for DNA via target-catalyzed hairpin assembly and the peroxidase-like catalytic of graphene/Au-NPs hybrids.

    PubMed

    Chen, Chaohui; Li, Ningxing; Lan, Jingwen; Ji, Xinghu; He, Zhike

    2016-01-01

    A target-catalyzed hairpin assembly (CHA) and graphene/Au-NPs hybrids-based platform has been developed for the determination of DNA. This new sensor not only avoided any labeling but also reduced the background signal. In the absence of target, the assembly of H1 and H2 couldn't be triggered. The catalytic activity of graphene/Au-NPs hybrids was inhibited by adsorption of H1 and H2, leading to the "inactive" hybrids unable to catalyze the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). However, with the addition of target DNA, the target-catalyzed hairpin assembly was initiated and produced plenty of H1-H2 duplex, which had a weak binding affinity with the graphene/Au-NPs. Thus, the protected interface of graphene/Au-NPs hybrids became active and catalyzed the oxidation reaction of TMB accompanied with a colorless to-blue color change. This approach exhibited good sensitivity and specificity for target DNA with a detection limit of 5.74 × 10(-11) M, and realized the assay of target DNA in human serum samples. Besides, this sensor could be further expanded to detect viruses or proteins by adapting the corresponding aptamers, showing great potential in biochemical detections.

  12. The effect of the shape of single, sub-ms voltage pulses on the rates of surface immobilization and hybridization of DNA.

    PubMed

    Cabeça, R; Rodrigues, M; Prazeres, D M F; Chu, V; Conde, J P

    2009-01-01

    Electric fields generated by single square and sinusoidal voltage pulses with amplitudes below 2 V were used to assist the covalent immobilization of single-stranded, thiolated DNA probes, onto a chemically functionalized SiO2 surface and to assist the specific hybridization of single-stranded DNA targets with immobilized complementary probes. The single-stranded immobilized DNA probes were either covalently immobilized (chemisorption) or electrostatically adsorbed (physisorption) to a chemically functionalized surface. Comparing the speed of electric field assisted immobilization and hybridization with the corresponding control reactions (without electric field), an increase of several orders of magnitude is observed, with the reaction timescaled down from 1 to 2 h to a range between 100 ns and 1 ms. The influence of the shape of the voltage pulse (square versus sinusoidal) and its duration were studied for both immobilization and hybridization reactions. The results show that pulsed electric fields are a useful tool to achieve temporal and spatial control of surface immobilization and hybridization reactions of DNA. PMID:19417254

  13. Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

    PubMed

    Nishiyama, Milton Yutaka; Ferreira, Savio Siqueira; Tang, Pei-Zhong; Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100-130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

  14. Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    PubMed Central

    Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation. PMID:25222706

  15. A multilevel ant colony optimization algorithm for classical and isothermic DNA sequencing by hybridization with multiplicity information available.

    PubMed

    Kwarciak, Kamil; Radom, Marcin; Formanowicz, Piotr

    2016-04-01

    The classical sequencing by hybridization takes into account a binary information about sequence composition. A given element from an oligonucleotide library is or is not a part of the target sequence. However, the DNA chip technology has been developed and it enables to receive a partial information about multiplicity of each oligonucleotide the analyzed sequence consist of. Currently, it is not possible to assess the exact data of such type but even partial information should be very useful. Two realistic multiplicity information models are taken into consideration in this paper. The first one, called "one and many" assumes that it is possible to obtain information if a given oligonucleotide occurs in a reconstructed sequence once or more than once. According to the second model, called "one, two and many", one is able to receive from biochemical experiment information if a given oligonucleotide is present in an analyzed sequence once, twice or at least three times. An ant colony optimization algorithm has been implemented to verify the above models and to compare with existing algorithms for sequencing by hybridization which utilize the additional information. The proposed algorithm solves the problem with any kind of hybridization errors. Computational experiment results confirm that using even the partial information about multiplicity leads to increased quality of reconstructed sequences. Moreover, they also show that the more precise model enables to obtain better solutions and the ant colony optimization algorithm outperforms the existing ones. Test data sets and the proposed ant colony optimization algorithm are available on: http://bioserver.cs.put.poznan.pl/download/ACO4mSBH.zip.

  16. Inorganic-Organic Hybrid Luminescent Binary Probe for DNA Detection Based on Spin-Forbidden Resonance Energy Transfer

    PubMed Central

    Marti, Angel A.; Puckett, Cindy A.; Dyer, Joanne; Stevens, Nathan; Jockusch, Steffen; Ju, Jingyue; Barton, Jacqueline K.; Turro, Nicholas J.

    2009-01-01

    We describe the design of new fluorescent binary probe sensors for DNA detection based on spin-forbidden resonance energy transfer (SF-RET). Binary probes consist of a donor and acceptor fluorophores that are attached to two different oligonucleotides and serve as resonance energy transfer (RET) donor-acceptor pair when hybridized to adjacent sites of a target sequence. In the absence of target, excitation of the donor results in fluorescence only from the donor, but when the probes hybridize to target, the fluorophores are brought into close proximity favoring RET, yielding fluorescence mainly from the acceptor fluorophore. These new binary probes use the metal complex Ru(bpy′)(DIP)22+ as the energy donor and an organic fluorophore (Cy5) as the energy acceptor. Energy transfer from the MLCT state of the Ru complex to singlet Cy5 is spin forbidden and produces a delayed fluorescence of Cy5. This paper demonstrate that fluorescence delay of Cy5 can be used to time resolve the emission of the probe from the intense fluorescence background of a model system for cellular background; this provides the reported system to overcome intense autofluorescence, an important and general advantage over “classical” spin-allowed steady-state probes. PMID:17592843

  17. An ultrasensitive DNA biosensor based on covalent immobilization of probe DNA on fern leaf-like α-Fe2O3 and chitosan Hybrid film using terephthalaldehyde as arm-linker.

    PubMed

    Xu, Biyan; Zheng, Delun; Qiu, Weiwei; Gao, Feng; Jiang, Shaoxiong; Wang, Qingxiang

    2015-10-15

    In this work, a novel electrochemical DNA biosensor has been developed based on the hybrid film of fern leaf-like α-Fe2O3 microparticles and chitosan (CS). The fern leaf-like α-Fe2O3 microparticles were synthesized via a facile template-free hydrothermal method, and their morphologies were characterized by X-ray diffraction, energy dispersive spectrometry, scanning electron microscope, and transmission electron microscope. Electrochemical characterization assays revealed that the hybrid film modified electrode had remarkable synergistic effects of the large accessible surface area and high electrical conductivity of semiconductive Fe2O3, and the good film stability of CS. Based on the rich amino groups on CS, the CS-Fe2O3 hybrid film was employed as a functional matrix for probe DNA immobilization using terephthalaldehyde (TPA) as a bifunctional arm-linker. The hybridization capacity of the developed biosensor was evaluated with electrochemical impedance spectroscopy (EIS) using [Fe(CN)6](3-/4-) as the indicating probe. A wide dynamic detection range from 1.0 × 10(-14) to 1.0 × 10(-10)M with ultralow detection limit of 5.6 × 10(-15)M was achieved for the target DNA. The hybridization selectivity experiments further revealed that the biosensor could discriminate fully complementary sequences from one-base mismatched, three-base mismatched, and non-complementary sequences. Moreover, the biosensor showed the advantage of good regeneration ability and reproducibility.

  18. An ultrasensitive DNA biosensor based on covalent immobilization of probe DNA on fern leaf-like α-Fe2O3 and chitosan Hybrid film using terephthalaldehyde as arm-linker.

    PubMed

    Xu, Biyan; Zheng, Delun; Qiu, Weiwei; Gao, Feng; Jiang, Shaoxiong; Wang, Qingxiang

    2015-10-15

    In this work, a novel electrochemical DNA biosensor has been developed based on the hybrid film of fern leaf-like α-Fe2O3 microparticles and chitosan (CS). The fern leaf-like α-Fe2O3 microparticles were synthesized via a facile template-free hydrothermal method, and their morphologies were characterized by X-ray diffraction, energy dispersive spectrometry, scanning electron microscope, and transmission electron microscope. Electrochemical characterization assays revealed that the hybrid film modified electrode had remarkable synergistic effects of the large accessible surface area and high electrical conductivity of semiconductive Fe2O3, and the good film stability of CS. Based on the rich amino groups on CS, the CS-Fe2O3 hybrid film was employed as a functional matrix for probe DNA immobilization using terephthalaldehyde (TPA) as a bifunctional arm-linker. The hybridization capacity of the developed biosensor was evaluated with electrochemical impedance spectroscopy (EIS) using [Fe(CN)6](3-/4-) as the indicating probe. A wide dynamic detection range from 1.0 × 10(-14) to 1.0 × 10(-10)M with ultralow detection limit of 5.6 × 10(-15)M was achieved for the target DNA. The hybridization selectivity experiments further revealed that the biosensor could discriminate fully complementary sequences from one-base mismatched, three-base mismatched, and non-complementary sequences. Moreover, the biosensor showed the advantage of good regeneration ability and reproducibility. PMID:25982725

  19. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

    PubMed

    Kato, Hirotomo; Cáceres, Abraham G; Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.

  20. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

    PubMed

    Kato, Hirotomo; Cáceres, Abraham G; Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area. PMID:26735142

  1. First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru

    PubMed Central

    Hashiguchi, Yoshihisa

    2016-01-01

    The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area. PMID:26735142

  2. Detection of Vibrio cholerae O1 and O139 in environmental waters of rural Bangladesh: a flow-cytometry-based field trial.

    PubMed

    Righetto, L; Zaman, R U; Mahmud, Z H; Bertuzzo, E; Mari, L; Casagrandi, R; Gatto, M; Islam, S; Rinaldo, A

    2015-08-01

    Presence of Vibrio cholerae serogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination of V. cholerae cells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance of V. cholerae O1. These results extend earlier studies where V. cholerae O1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds' host populations of V. cholerae are able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations of V. cholerae concentrations with environmental factors and the spatial distribution of V. cholerae populations are also discussed. PMID:25496520

  3. Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development.

    PubMed

    Quadrini, Karen J; Hegelund, Anne Charlotte; Cortes, Kasia E; Xue, Chengsen; Kennelly, Susan M; Ji, Hong; Högerkorp, Carl-Magnus; Mc Closkey, Thomas W

    2016-03-01

    The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial. PMID:26084468

  4. A flow cytometry-based immuno-titration assay for rapid and accurate titer determination of modified vaccinia Ankara virus vectors.

    PubMed

    Li, Zengji; Ling, Loni; Liu, Xiaohui; Laus, Reiner; Delcayre, Alain

    2010-10-01

    A flow cytometry-based immuno-titration titer assay was established to determine infectious unit (IU) and transducing unit (TU) of modified vaccinia Ankara (MVA) virus vectors. This titration method enumerates infected cells by measuring the expression of viral protein for IU and transgene protein for TU in individual cells after staining with fluorophore-conjugated antibodies. It presents many advantages over standard virus titration approaches, such as TCID(50) or plaque assay, for its convenience, rapidity and accuracy as illustrated by excellent assay linearity and reproducibility. Importantly, the IU and the TCID(50) assays generated similar batch-specific titer values when testing varied MVA-derived virus preparations. Assay development revealed that the post-infection time at which viral protein expression is evaluated, host cell type, and blocking the formation and release of progeny virion with nocodazole, an anti-microtubule agent or rifampin, a specific vaccinia virus assembly inhibitor, are critical parameters for the precision, robustness, and accuracy of IU titer determination. An added advantage of this assay is that it enables the concurrent determination of IU and transducing units (TU) by measuring the expression of a transgene product when testing recombinant viruses. The latter was demonstrated using a MVA vector carrying a human HER-2 gene fragment as model. Hence, this assay is very versatile in that it can be used to determine IU as well as multiple TU titers simultaneously. Furthermore, it can readily be adapted to other poxvirus vectors.

  5. Titration of varicella-zoster virus DNA in throat swabs from varicella patients by combined use of PCR and microplate hybridization.

    PubMed

    Hondo, R; Ito, S; Inouye, S

    1995-01-01

    We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.

  6. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.

    PubMed

    Mahfouz, Magdy M; Li, Lixin; Shamimuzzaman, Md; Wibowo, Anjar; Fang, Xiaoyun; Zhu, Jian-Kang

    2011-02-01

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  7. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    PubMed

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  8. Geographic divergence of "Sulfolobus islandicus" strains assessed by genomic analyses including electronic DNA hybridization confirms they are geovars.

    PubMed

    Zuo, Guanghong; Hao, Bailin; Staley, James T

    2014-02-01

    Ten well-annotated genomes of "Sulfolobus islandicus" strains from different geographic locations have been released at the NCBI database. Whole genome based composition vector trees indicate that these strains show the same branching patterns as originally reported by multi-locus sequence analysis. To determine whether the ten strains meet the criteria for separate species, DNA-DNA hybridization (DDH) was performed in silico. DDH values of strains from the same geographic location, i.e., Iceland, Kamchatka and North America, ranged from 82.4 to 95.4 %, clearly qualifying them as members of the same species. The lowest DDH values found between locations ranged from 75.5 to 76.6 %, which exceed the 70 % DDH threshold for a species thereby indicating they are all members of the same species based on the currently accepted definition. The clear divergences of strains from the different geographic locations are sufficiently great to consider them as separate geovars. "S. islandicus" has not yet been validly named and a type strain has not been deposited in culture collections. We urgently recommend that those who study the organism fulfill the criteria of the International Code of Nomenclature of Bacteria in order to designate a type strain and to identify and deposit related strains of this species to make them available to the broader scientific community.

  9. Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA–DNA hybrids

    PubMed Central

    Abraham, Karan J.; Chan, Janet N.Y.; Salvi, Jayesh S.; Ho, Brandon; Hall, Amanda; Vidya, Elva; Guo, Ru; Killackey, Samuel A.; Liu, Nancy; Lee, Jeffrey E.; Brown, Grant W.; Mekhail, Karim

    2016-01-01

    Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg2+) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg2+ is unknown. Here, we show that Mg2+, acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA–DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg2+ transporters Alr1/2 act via Mg2+-sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg2+ in a process that is dependent on the TRPM7 Mg2+ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg2+ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes. PMID:27574117

  10. DNA-LCEB: a high-capacity and mutation-resistant DNA data-hiding approach by employing encryption, error correcting codes, and hybrid twofold and fourfold codon-based strategy for synonymous substitution in amino acids.

    PubMed

    Hafeez, Ibbad; Khan, Asifullah; Qadir, Abdul

    2014-11-01

    Data-hiding in deoxyribonucleic acid (DNA) sequences can be used to develop an organic memory and to track parent genes in an offspring as well as in genetically modified organism. However, the main concerns regarding data-hiding in DNA sequences are the survival of organism and successful extraction of watermark from DNA. This implies that the organism should live and reproduce without any functional disorder even in the presence of the embedded data. Consequently, performing synonymous substitution in amino acids for watermarking becomes a primary option. In this regard, a hybrid watermark embedding strategy that employs synonymous substitution in both twofold and fourfold codons of amino acids is proposed. This work thus presents a high-capacity and mutation-resistant watermarking technique, DNA-LCEB, for hiding secret information in DNA of living organisms. By employing the different types of synonymous codons of amino acids, the data storage capacity has been significantly increased. It is further observed that the proposed DNA-LCEB employing a combination of synonymous substitution, lossless compression, encryption, and Bose-Chaudary-Hocquenghem coding is secure and performs better in terms of both capacity and robustness compared to existing DNA data-hiding schemes. The proposed DNA-LCEB is tested against different mutations, including silent, miss-sense, and non-sense mutations, and provides substantial improvement in terms of mutation detection/correction rate and bits per nucleotide. A web application for DNA-LCEB is available at http://111.68.99.218/DNA-LCEB.

  11. A sandwich-hybridization assay for simultaneous determination of HIV and tuberculosis DNA targets based on signal amplification by quantum dots-PowerVision™ polymer coding nanotracers.

    PubMed

    Yan, Zhongdan; Gan, Ning; Zhang, Huairong; Wang, De; Qiao, Li; Cao, Yuting; Li, Tianhua; Hu, Futao

    2015-09-15

    A novel sandwich-hybridization assay for simultaneous electrochemical detection of multiple DNA targets related to human immune deficiency virus (HIV) and tuberculosis (TB) was developed based on the different quantum dots-PowerVision(TM) polymer nanotracers. The polymer nanotracers were respectively fabricated by immobilizing SH-labeled oligonucleotides (s-HIV or s-TB), which can partially hybrid with virus DNA (HIV or TB), on gold nanoparticles (Au NPs) and then modified with PowerVision(TM) (PV) polymer-encapsulated quantum dots (CdS or PbS) as signal tags. PV is a dendrimer enzyme linked polymer, which can immobilize abundant QDs to amplify the stripping voltammetry signals from the metal ions (Pb or Cd). The capture probes were prepared through the immobilization of SH-labeled oligonucleotides, which can complementary with HIV and TB DNA, on the magnetic Fe3O4@Au (GMPs) beads. After sandwich-hybridization, the polymer nanotracers together with HIV and TB DNA targets were simultaneously introduced onto the surface of GMPs. Then the two encoding metal ions (Cd(2+) and Pb(2+)) were used to differentiate two viruses DNA due to the different subsequent anodic stripping voltammetric peaks at -0.84 V (Cd) and -0.61 V (Pb). Because of the excellent signal amplification of the polymer nanotracers and the great specificity of DNA targets, this assay could detect targets DNA as low as 0.2 femtomolar and exhibited excellent selectivity with the dynamitic range from 0.5 fM to 500 pM. Those results demonstrated that this electrochemical coding assay has great potential in applications for screening more viruses DNA while changing the probes.

  12. An experimentally-informed coarse-grained 3-Site-Per-Nucleotide model of DNA: structure, thermodynamics, and dynamics of hybridization.

    PubMed

    Hinckley, Daniel M; Freeman, Gordon S; Whitmer, Jonathan K; de Pablo, Juan J

    2013-10-14

    A new 3-Site-Per-Nucleotide coarse-grained model for DNA is presented. The model includes anisotropic potentials between bases involved in base stacking and base pair interactions that enable the description of relevant structural properties, including the major and minor grooves. In an improvement over available coarse-grained models, the correct persistence length is recovered for both ssDNA and dsDNA, allowing for simulation of non-canonical structures such as hairpins. DNA melting temperatures, measured for duplexes and hairpins by integrating over free energy surfaces generated using metadynamics simulations, are shown to be in quantitative agreement with experiment for a variety of sequences and conditions. Hybridization rate constants, calculated using forward-flux sampling, are also shown to be in good agreement with experiment. The coarse-grained model presented here is suitable for use in biological and engineering applications, including nucleosome positioning and DNA-templated engineering. PMID:24116642

  13. Identification of rat lung – prominent genes by a parallel DNA microarray hybridization

    PubMed Central

    Chen, Zhongming; Chen, Jiwang; Weng, Tingting; Jin, Nili; Liu, Lin

    2006-01-01

    Background The comparison of organ transcriptomes is an important strategy for understanding gene functions. In the present study, we attempted to identify lung-prominent genes by comparing the normal transcriptomes of rat lung, heart, kidney, liver, spleen, and brain. To increase the efficiency and reproducibility, we first developed a novel parallel hybridization system, in which 6 samples could be hybridized onto a single slide at the same time. Results We identified the genes prominently expressed in the lung (147) or co-expressed in lung-heart (23), lung-liver (37), lung-spleen (203), and lung-kidney (98). The known functions of the lung-prominent genes mainly fell into 5 categories: ligand binding, signal transducer, cell communication, development, and metabolism. Real-time PCR confirmed 13 lung-prominent genes, including 5 genes that have not been investigated in the lung, vitamin D-dependent calcium binding protein (Calb3), mitogen activated protein kinase 13 (Mapk13), solute carrier family 29 transporters, member 1 (Slc29a1), corticotropin releasing hormone receptor (Crhr1), and lipocalin 2 (Lcn2). Conclusion The lung-prominent genes identified in this study may provide an important clue for further investigation of pulmonary functions. PMID:16533406

  14. Label-free electrical detection of DNA hybridization using carbon nanotubes and graphene

    PubMed Central

    Fu, Dongliang; Li, Lain-Jong

    2010-01-01

    The interface between biosystems and nanomaterials is emerging for detection of various biomolecules and subtle cellular activities. In particular, the development of cost-effective and sequence-selective DNA detection is urgent for the diagnosis of genetic or pathogenic diseases. Graphene-based nanocarbon materials, such as carbon nanotubes and thin graphene layers, have been employed as biosensors because they are biocompatible, extraordinarily sensitive, and promising for large-area detection. Electrical and label-free detection of DNA can be achieved by monitoring the conductance change of devices fabricated from these carbon materials. Here, the recent advances in this research area are briefly reviewed. The key issues and perspectives of future development are also discussed. PMID:22110861

  15. Incorporation of cyclic azobenzene into oligodeoxynucleotides for the photo-regulation of DNA hybridization.

    PubMed

    Eljabu, Fatma; Dhruval, Joshi; Yan, Hongbin

    2015-12-01

    Cyclic azobenzene carboxylic acid was synthesized using a shortened route. After reaction with D-threolinol, the resulting cyclic azobenzene-D-threolinol (cAB-Thr) building block was transformed into the corresponding DMTr-protected phosphoramidite, and incorporated into oligodeoxynucleotides at various positions and frequencies by solid phase synthesis. The melting temperatures of these modified oligonucleotides were determined by UV spectrometry. Photo-regulation of cAB-Thr-modified oligonucleotides with their complementary sequence was evaluated by Fluorescence Resonance Energy Transfer experiments using a fluorescein-Black Hole Quencher pair. Results suggest that while cis-cAB destabilizes DNA duplexes, trans-cAB can be accommodated in double stranded DNA.

  16. Incorporation of cyclic azobenzene into oligodeoxynucleotides for the photo-regulation of DNA hybridization.

    PubMed

    Eljabu, Fatma; Dhruval, Joshi; Yan, Hongbin

    2015-12-01

    Cyclic azobenzene carboxylic acid was synthesized using a shortened route. After reaction with D-threolinol, the resulting cyclic azobenzene-D-threolinol (cAB-Thr) building block was transformed into the corresponding DMTr-protected phosphoramidite, and incorporated into oligodeoxynucleotides at various positions and frequencies by solid phase synthesis. The melting temperatures of these modified oligonucleotides were determined by UV spectrometry. Photo-regulation of cAB-Thr-modified oligonucleotides with their complementary sequence was evaluated by Fluorescence Resonance Energy Transfer experiments using a fluorescein-Black Hole Quencher pair. Results suggest that while cis-cAB destabilizes DNA duplexes, trans-cAB can be accommodated in double stranded DNA. PMID:26592170

  17. Synthesis of Ag(2) S-Ag nanoprisms and their use as DNA hybridization probes.

    PubMed

    Liu, Bing; Ma, Zhanfang

    2011-06-01

    A simple synthetic route to prepare Ag(2) S-Ag nanoprisms consists of the facile addition of Na(2) S to a solution of triangular Ag nanoprisms. The resulting Ag(2) S-Ag nanoparticles are more stable in solution than the original Ag nanoprisms, and two surface plasmon resonance (SPR) bands of the original Ag nanoprisms still remain. In addition, the SPR bands of the Ag(2) S-Ag nanoprisms are tunable over a wide range. The Ag(2) S-Ag nanoprisms can be directly bioconjugated via well-established stable Ag(2) S surface chemistry with readily available sulfur coupling agents. The nanoprisms are used in the hybridization of functionalized oligonucleotides, and show promise as probes for future biosensing applications. PMID:21538868

  18. Immune and histopathologic responses of DNA-vaccinated hybrid striped bass Morone saxatilis x M. chrysops after acute Mycobacterium marinum infection.

    PubMed

    Pasnik, David J; Smith, Stephen A

    2006-11-21

    The post-challenge immune and histopathologic responses of hybrid striped bass vaccinated with a DNA vaccine encoding the Mycobacterium marinum Ag85A gene and subsequently challenged with M. marinum were investigated. Juvenile hybrid striped bass Morone saxatilis x M. chrysops were injected intramuscularly with 25 or 50 microg DNA plasmid and developed significant specific protective responses to live bacterial challenge 120 d post-vaccination. Both vaccine groups demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups 14 d after challenge with approximately 8 x 10(5) cfu M. marinum g(-1) fish body wt. The vaccine groups also developed more rapidly and significantly increased antibody and lymphoproliferative responses post-challenge compared to control groups, and these post-challenge immune responses appear to be vital against M. marinum infection in vaccinated hybrid striped bass. No significant differences in immune responses were recognized between the 25 and 50 microg vaccination groups, and these groups eventually experienced mortalities, splenic bacterial counts, and granuloma formation 28 d post-challenge comparable to those of the control groups at 14 d post-challenge. Therefore, vaccination of hybrid striped bass with a DNA vaccine encoding the M. marinum Ag85A gene provided significant but limited duration of protection against an acute high-dose M. marinum challenge.

  19. The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6.

    PubMed

    Phillips, Damilola D; Garboczi, David N; Singh, Kavita; Hu, Zonglin; Leppla, Stephen H; Leysath, Clinton E

    2013-08-01

    The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

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

    PubMed

    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.

  1. Highly sensitive gold nanoparticles-based optical sensing of DNA hybridization using bis(8-hydroxyquinoline-5-solphonate)cerium(III) chloride as a novel fluorescence probe.

    PubMed

    Shamsipur, Mojtaba; Memari, Zahra; Ganjali, Mohammad Reza; Norouzi, Parviz; Faridbod, Farnoush

    2016-01-25

    A simple and sensitive method for the detection of DNA hybridization in a homogeneous format was developed, using bis(8-hydroxyquinoline-5-solphonate)cerium(III) chloride (Ce(QS)2Cl) as a novel fluorescent probe. The method is based on fluorescence quenching by gold nanoparticles used as both nanoscafolds for the immobilization of the probe DNA sequence, which is related to Alicyclobacillus acidophilus strain TA-67 16S ribosomal RNA, and nanoquenchers of the Ce(QS)2Cl probe. The probe DNA-functionalized GNPs were synthesized by derivatizing the colloidal gold nanoparticles solution with 3-thiolated 16-base oligonucleotides. Addition of sequence-specific target DNAs (16 bases) into the mixture containing probe DNA-functionalized GNPs and fluorescent probe lead to the quenching of Ce(QS)2Cl fluorescence at 360 nm (λex=270 nm), due to DNA hybridization, the resulting quenched intensity being proportional to the concentration of target DNA. Under optimal conditions of pH 7.4 and Ce(QS)2Cl concentration of 1.0 × 10(-7) M, the linear dynamic range found to be 1.0 × 10(-10)-3.0 × 10(-8) M DNA, with a limit of detection of 7.0 × 10(-11) M. The interaction mechanism for the binding of Ce(QS)2Cl to DNA was studied in detail, and results proved that the interaction mode between Ce(QS)2Cl and DNA is groove binding, with a binding constant of 1.0 × 10(5) M(-1).

  2. An investigation into the influence of secondary structures for DNA hybridization using surface plasmon resonance and surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Yih, J.-N.; Chiu, K.-C.; Chien, F.-C.; Chen, W.-Y.; Chen, S.-J.

    2006-02-01

    This study utilizes a surface plasmon resonance (SPR) biosensing to investigate the influence of secondary structures on the DNA hybridization and a surface-enhanced Raman scattering (SERS) spectrum to yield analytical data regarding the structure of the oligonucleotides. It is found that the SPR angular shifts associated with the three pairs of 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 °C to 45 °C reduces secondary structure effects. On the hybridization with mixture target oligonucleotides, the SPR results demonstrate that secondary structures interfere significantly. Although the kinetics of biomolecular interaction analysis is performed by using SPR sensor, the structural information of the oligonucleotides can not observed directly. The SERS spectrum provides the structural information of the oligonucleotides with silver colloidal nanoparticles adapted as a Raman active substrate. Also, the detection limit of the DNA Raman signal has been successfully improved to reach sub-micro molarity of DNA concentration.

  3. Allozyme and mitochondrial DNA analysis of a hybrid zone between white-tailed deer and mule deer (Odocoileus) in west Texas.

    PubMed

    Ballinger, S W; Blankenship, L H; Bickham, J W; Carr, S M

    1992-02-01

    Thirty allozyme loci and 35 mitochondrial DNA (mtDNA) restriction sites were examined in 24 white-tailed deer and 46 mule deer from a hybrid zone in West Texas. A common mtDNA genotype is shared by all of the mule deer with 67% of the white-tailed deer. At the albumin locus, 13% of the white-tailed deer and 24% of the mule deer are heterozygous, sharing alleles that are otherwise species-specific in allopatric populations; 7% of the mule deer are homozygous for the allele that is characteristic of allopatric white-tailed deer. Gene flow appears to have been bidirectional, with greater genetic introgression into mule deer. The mtDNA data suggest that matings between white-tailed and mule deer have occurred in the past. Despite evidence of genetic introgression, analysis of multilocus genotypes indicates that none of the deer examined is an F1 hybrid. Production of such hybrids appears to be generally uncommon in North American deer; management plans that assume otherwise should be reconsidered.

  4. Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

    PubMed Central

    Kaufman, S L; Gallo, R C; Miller, N R

    1979-01-01

    An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA. Images PMID:224220

  5. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  6. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  7. Comparison of pp65 antigenemia, quantitative PCR and DNA hybrid capture for detection of cytomegalovirus in transplant recipients and AIDS patients.

    PubMed

    Mhiri, Leila; Kaabi, Belhassen; Houimel, Mehdi; Arrouji, Zakia; Slim, Amine

    2007-07-01

    The cytomegalovirus (CMV) antigenemia assay has been used frequently for rapid diagnosis of CMV infection, and antigenemia threshold values are recommended for triggering preemptive therapy. Hybrid capture of CMV's DNA and quantitative polymerase chain reaction (qPCR) are increasingly being adopted for early detection of CMV. The performance of the antigenemia assay, qPCR in plasma and hybrid capture in leukocytes were compared in 110 immunocompromised patients (38 bone-marrow transplants, 50 renal transplants and 22 AIDS patients). The most sensitive test was hybrid capture for transplants, while antigenemia and the qPCR showed similar performance for patients with AIDS. QPCR and hybrid capture thresholds requiring antiviral therapy were calculated using a receiver-operating-characteristic curve for antigenemia values corresponding to 2 positive cells for bone-marrow transplants and to 10 positive cells for renal transplants and AIDS patients. These threshold values varied with the group of patients considered, with corresponding sensitivities higher than 86% and specificities higher than 76% for hybrid capture, and sensitivities higher than 61% and specificities higher than 75% for qPCR in plasma. Hybrid capture in leukocytes can substitute for antigenemia in the case of transplants, and qPCR in plasma can substitute for it in the case of AIDS patients.

  8. An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening.

    PubMed

    Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

    2006-01-01

    We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.

  9. Some properties of site-specific nickase BspD6I and the possibility of its use in hybridization analysis of DNA.

    PubMed

    Zheleznaya, L A; Perevyazova, T A; Zheleznyakova, E N; Matvienko, N I

    2002-04-01

    A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55 degrees C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.

  10. [Genetic variability of sable Martes zibellina L., pine marten M. martes L., and their hybrids in Western Siberia: protein and DNA polymorphism].

    PubMed

    Zhigileva, O N; Politov, D V; Golovacheva, I M; Petrovicheva, S V

    2014-05-01

    Using four types of markers, the genetic variability of sable and pine marten inhabiting Western Siberia was examined. Izoenzyme and restriction endonuclease analysis of the mtDNA cytochrome b gene fragment, as well as the ISSR-PCR and analysis of microsatellite variation, revealed a low differentiation level of sable and pine marten and confirmed the hybrid origin of atypical representatives of these species. The hybrids were characterized by an increased heterozygosity level and were genetically closer to sable than to pine marten. In atypical martens, the presence of mtDNA haplotypes of eastern sable was identified. This could be the consequence of the reintroduction of the Barguzin sable in the 20th century. In Western Siberia, the introgression of genes between sable and pine marten was massive and symmetrical. It apparently occurred in the past and continues in the present.

  11. Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis

    NASA Astrophysics Data System (ADS)

    Bao, Chenchen; Conde, João; Curtin, James; Artzi, Natalie; Tian, Furong; Cui, Daxiang

    2015-07-01

    Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.

  12. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. 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 will generate 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.

  13. Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array

    PubMed Central

    Anthony, R. M.; Brown, T. J.; French, G. L.

    2000-01-01

    The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost. PMID:10655385

  14. The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.

    PubMed

    Molodtsov, Vadim; Anikin, Michael; McAllister, William T

    2014-09-01

    Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway. PMID:24976131

  15. Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis.

    PubMed

    Bao, Chenchen; Conde, João; Curtin, James; Artzi, Natalie; Tian, Furong; Cui, Daxiang

    2015-01-01

    Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases. PMID:26189409

  16. Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis

    PubMed Central

    Bao, Chenchen; Conde, João; Curtin, James; Artzi, Natalie; Tian, Furong; Cui, Daxiang

    2015-01-01

    Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases. PMID:26189409

  17. Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy.

    PubMed

    Lizard, G; Chignol, M C; Roignot, P; Souchier, C; Chardonnet, Y; Schmitt, D

    1997-07-01

    Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.

  18. Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus

    SciTech Connect

    Uhl, G.R.; Zingg, H.H.; Habener, J.F.

    1985-08-01

    Hybridizable vasopressin mRNA may be quantitatively localized in situ in sections from rat hypothalamus. Radiolabeled oligonucleotide cDNA probes, synthesized by chemical and enzymatic means, provide strong hybridization in zones known to contain vasopressin cell bodies. Multiple single-stranded /sup 32/P-, /sup 35/S-, or /sup 3/H-labeled oligonucleotides demonstrate localized hybridization that increases as probes are lengthened from 8 to 75 bases. Competition studies, RNase experiments, anatomic localization, and use of multiple probes all support hybridization specificity. An approximate doubling of hybridizable mRNA in both supraoptic and paraventricular nuclei can be detected with dehydration of the animals. Hybridizable mRNA densities are virtually normal in hypothalamic nuclei of Brattleboro rats given free access to water. These methods can provide insight into regional mRNA dynamics and may reflect functional activity of peptidergic neurons.

  19. Chromosome mapping of 18S rDNA and 5S rDNA by dual-color fluorescence in situ hybridization in the half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Jiang, L; Jiang, J; Liu, J; Yuan, J; Chen, Y; Zhang, Q; Wang, X

    2014-12-18

    Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture flatfish in China. Cytogenetic analysis has revealed that its sex determination system is female heterogametic (ZZ/ZW). The W chromosome is morphologically larger and has been considered evolutionarily younger than any other chromosome in the set. However, the genetic origin and evolution process of this neo-chromosome remains unclear. In this study, 2 tandem arrays of rRNA genes were chosen to address this question. Both the major rDNA (18S rDNA) and the minor rDNA (5S rDNA) were located on the C. semilaevis chromosomes by fluorescence in situ hybridization (FISH). Six 18S rDNA signals were observed on the centromeric regions of 3 pairs of autosomes in both males and females. In females, there was an additional 18S rDNA signal mapping to the telomeric region of the W chromosome long arm. With respect to the 5S rDNA, 12 signals were mapped to the centromeric regions of six pairs of autosomes. Two-color FISH further confirmed that the two pairs of the 5S rDNA signals were correspondingly located at the same positions of the same autosomes as those of the 18S rDNA signals. These results allowed us to speculate about the evolution process of the W chromosome. Chromosome fusions and repetitive sequence accumulations might have occurred in C. semilaevis. The synteny and non-synteny of C. semilaevis 18S rDNA and 5S rDNA might imply the original and evolutionary characteristics of this species. These findings will facilitate studies on karyotype evolution of the order Pleuronectiformes.

  20. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    SciTech Connect

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; Desireddy, Anil; Artyushkova, Kateryna; Boncella, Amy E.; Atanassov, Plamen; Martinez, Jennifer S.

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

  1. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    DOE PAGES

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; Desireddy, Anil; Artyushkova, Kateryna; Boncella, Amy E.; Atanassov, Plamen; Martinez, Jennifer S.

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonicmore » gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.« less

  2. Novel hybrid maturases in unstable pseudorevertants of maturaseless mutants of yeast mitochondrial DNA.

    PubMed Central

    Anziano, P Q; Moran, J V; Gerber, D; Perlman, P S

    1990-01-01

    Unstable pseudorevertants of mitochondrial mutants of Saccharomyces cerevisiae lacking the maturase function encoded by the fourth intron of the cytochrome b gene (bI4) were isolated. They were found to be heteroplasmic cells owing their regained ability to respire (and grow on glycerol medium) to the presence of a rearranged (rho-) mtDNA that contains an in-frame fusion of the reading frames of the group I introns bI4 and intron 4 alpha of the coxl gene encoding subunit I of cytochrome c oxidase (aI4 alpha). The products of those gene fusions suppress the bI4 maturase deficiency still present in those heteroplasmic cells. Similar heteroplasmic pseudorevertants of a group II maturaseless mutant of the first intron of the coxI gene were characterized; they result from partial deletion of the coxI gene that fuses the reading frames of introns 1 and 2. These heteroplasms provide independent support for the existence of RNA maturases encoded by group I and group II introns. Also, since the petite/mit- heteroplasms arise spontaneously at very high frequencies they provide a system that can be used to obtain mutants unable to form or maintain heteroplasmic cells. Images PMID:1972561

  3. COMPARISON OF COMPARATIVE GENOMIC HYBRIDIZATIONS TECHNOLOGIES ACROSS MICROARRAY PLATFORMS

    EPA Science Inventory

    Comparative Genomic Hybridization (CGH) measures DNA copy number differences between a reference genome and a test genome. The DNA samples are differentially labeled and hybridized to an immobilized substrate. In early CGH experiments, the DNA targets were hybridized to metaphase...

  4. Evaluation of DNA colony hybridization and real-time PCR for detection of Vibrio parahaemolyticus and Vibrio vulnificus in postharvest-processed oysters.

    PubMed

    Jones, Jessica L; Noe, Kathy E; Byars, Robin; Depaola, Angelo

    2009-10-01

    The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.

  5. Effects of Concentration and Temperature on DNA Hybridization by Two Closely Related Sequences via Large-Scale Coarse-Grained Simulations.

    PubMed

    Markegard, Cade B; Gallivan, Cameron P; Cheng, Darrell D; Nguyen, Hung D

    2016-08-18

    A newly developed coarse-grained model called BioModi is utilized to elucidate the effects of temperature and concentration on DNA hybridization in self-assembly. Large-scale simulations demonstrate that complementary strands of either the tetrablock sequence or randomized sequence with equivalent number of cytosine or guanine nucleotides can form completely hybridized double helices. Even though the end states are the same for the two sequences, there exist multiple kinetic pathways that are populated with a wider range of transient aggregates of different sizes in the system of random sequences compared to that of the tetrablock sequence. The ability of these aggregates to undergo the strand displacement mechanism to form only double helices depends upon the temperature and DNA