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Sample records for specific dna hybridization

  1. Sensitivity, Specificity, and the Hybridization Isotherms of DNA Chips

    PubMed Central

    Halperin, A.; Buhot, A.; Zhulina, E.B.

    2004-01-01

    Competitive hybridization, at the surface and in the bulk, lowers the sensitivity of DNA chips. Competitive surface hybridization occurs when different targets can hybridize with the same probe. Competitive bulk hybridization takes place when the targets can hybridize with free complementary chains in the solution. The effects of competitive hybridization on the thermodynamically attainable performance of DNA chips are quantified in terms of the hybridization isotherms of the spots. These relate the equilibrium degree of the hybridization to the bulk composition. The hybridization isotherm emerges as a Langmuir isotherm modified for electrostatic interactions within the probe layer. The sensitivity of the assay in equilibrium is directly related to the slope of the isotherm. A simpler description is possible, in terms of c50 values specifying the bulk composition corresponding to 50% hybridization at the surface. The effects of competitive hybridization are important for the quantitative analysis of DNA chip results, especially when used to study point mutations. PMID:14747310

  2. 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

  3. 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

  4. A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

    PubMed Central

    Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

    2011-01-01

    In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

  5. 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

  6. 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

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

    PubMed

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

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library. PMID:27108327

  8. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    PubMed

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  9. A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

    PubMed

    Finerty, P J; Bass, B L

    1997-08-15

    Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix. PMID:9268652

  10. 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

  11. 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

  12. 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.

  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. 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. PMID:9196184

  15. Chromosome painting in plants: in situ hybridization with a DNA probe from a specific microdissected chromosome arm of common wheat.

    PubMed Central

    Vega, M; Abbo, S; Feldman, M; Levy, A A

    1994-01-01

    We report here on the successful painting of a specific plant chromosome within its own genome. Isochromosomes for the long arm of chromosome 5 of the wheat B genome (5BL) were microdissected from first meiotic metaphase spreads of a monoisosomic 5BL line of the common wheat Triticum aestivum cv. Chinese Spring. The dissected isochromosomes were amplified by degenerate oligonucleotide-primed PCR in a single tube reaction. The amplified DNA was used as a complex probe mixture for fluorescent in situ hybridization on first meiotic metaphase spreads of lines carrying 5BL as a distinctive marker. Hybridization signals were observed, specifically, along the entire 5BL. In some of the cells, labeling was also detected in two bivalents, presumably those of the 5B "homoeologues" (partial homologues) found in common wheat (5A and 5D). The probe also revealed discrete domains in tapetal nuclei at interphase, further supporting the probe's high specificity. These data suggest that chromosome and homoeologous group-specific sequences are more abundant in 5BL than genome-specific sequences. Chromosome-painting probes, such as the one described here for 5BL, can facilitate the study of chromosome evolution in polyploid wheat. Images PMID:7991581

  16. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  17. 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...

  18. Development of genome-specific 5S rDNA markers in Brassica and related species for hybrid testing.

    PubMed

    La Mura, Maurizio; Norris, Carol; Sporle, Sue; Jayaweera, Dasuni; Greenland, Andy; Lee, David

    2010-08-01

    The Brassicaceae are targets for DNA manipulation to modify oil content and composition. However, any strategy for creating novel products using genetic modification or traditional breeding must take into account the potential for hybridization with other Brassica species, many of which are important sources of edible oils. In this study we have tested Brassica carinata, a possible target for oil modification, to establish whether it can cross with other Brassica species and related genera, and we have developed molecular DNA assays to confirm hybridization. PMID:20725152

  19. COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC GENETIC MARKERS IN CATTLE FECAL SAMPLES - ABSTRACT

    EPA Science Inventory

    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 met...

  20. COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC MICROBIAL GENETIC MARKERS IN COW FECAL SAMPLES

    EPA Science Inventory

    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 met...

  1. Quantum dot-based isothermal chain elongation for fluorescence detection of specific DNA sequences via template-dependent surface-hybridization.

    PubMed

    Song, Wenqing; Lau, Choiwan; Lu, Jianzhong

    2012-04-01

    A new quantum dot-based method to detect specific sequences of DNA is proposed. The capture and reporter probes do not hybridize to each other, but in the presence of a template they can anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the 5' end of the template target DNA remains free to hybridize with another reporter. In this way, each capture DNA is an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This forms a superstructure, enhances base stacking, and produces a strong fluorescent signal. The introduction of T4 DNA ligase further stabilizes the superstructure and greatly increases the fluorescence intensity, and the detection limit is as low as 10 fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap. This simple technique is promising for improving medical diagnosis and treatment. PMID:22343986

  2. Chromosome specific repetitive DNA sequences

    DOEpatents

    Moyzis, Robert K.; Meyne, Julianne

    1991-01-01

    A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

  3. 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

  4. Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination.

    PubMed

    Futai, Kazuki; Sumaoka, Jun; Komiyama, Makoto

    2016-05-01

    By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids. PMID:27057646

  5. DNA probe specific for Legionella pneumophila.

    PubMed Central

    Grimont, P A; Grimont, F; Desplaces, N; Tchen, P

    1985-01-01

    A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila. Images PMID:3980693

  6. Sequencing mitochondrial DNA polymorphisms by hybridization

    SciTech Connect

    Chee, M.S.; Lockhart, D.J.; Hubbell, E.

    1994-09-01

    We have investigated the use of DNA chips for genetic analysis, using human mitochondrial DNA (mtDNA) as a model. The DNA chips are made up of ordered arrays of DNA oligonucleotide probes, synthesized on a glass substrate using photolithographic techniques. The synthesis site for each different probe is specifically addressed by illumination of the substrate through a photolithographic mask, achieving selective deprotection Nucleoside phosphoramidites bearing photolabile protecting groups are coupled only to exposed sites. Repeated cycles of deprotection and coupling generate all the probes in parallel. The set of 4{sup N} N-mer probes can be synthesized in only 4N steps. Any subset can be synthesized in 4N steps. Any subset can be synthesized in 4N or fewer steps. Sequences amplified from the D-loop region of human mitochondrial DNA (mtDNA) were fluorescently labelled and hybridized to DNA chips containing probes specific for mtDNA. Each nucleotide of a 1.3 kb region spanning the D loop is represented by four probes on the chip. Each probe has a different base at the position of interest: together they comprise a set of A, C, G and T probes which are otherwise identical. In principle, only one probe-target hybrid will be a perfect match. The other three will be single base mismatches. Fluorescence imaging of the hybridized chip allows quantification of hybridization signals. Heterozygous mixtures of sequences can also be characterized. We have developed software to quantitate and interpret the hybridization signals, and to call the sequence automatically. Results of sequence analysis of human mtDNAs will be presented.

  7. 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

  8. In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

    PubMed Central

    Alexandersen, S; Bloom, M E; Wolfinbarger, J; Race, R E

    1987-01-01

    Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis. Images PMID:3037104

  9. 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

  10. 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.

  11. Signal enhancement in electronic detection of DNA hybridization

    NASA Astrophysics Data System (ADS)

    Gentil, C.; Philippin, G.; Bockelmann, U.

    2007-01-01

    Electronic detection of the specific recognition between complementary DNA sequences is investigated. DNA probes are immobilized at different lateral positions on a Poly( L -lysine)-coated surface of an integrated silicon transistor array. Hybridization and field effect detection are done with the solid surface immersed in electrolyte solutions. Differential measurements are performed, where DNA hybridization leads to surface potential shifts between the transistors of the array. We experimentally show that these differential signals of hybridization can be enhanced significantly by changing the salt concentration between hybridization and detection.

  12. Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

    PubMed

    Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

    2014-01-01

    Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. PMID:24956106

  13. Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

    PubMed Central

    Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

    2010-01-01

    We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

  14. 1997 hybrid electric vehicle specifications

    SciTech Connect

    Sluder, S.; Larsen, R.; Duoba, M.

    1996-10-01

    The US DOE sponsors Advanced Vehicle Technology competitions to help educate the public and advance new vehicle technologies. For several years, DOE has provided financial and technical support for the American Tour de Sol. This event showcases electric and hybrid electric vehicles in a road rally across portions of the northeastern United States. The specifications contained in this technical memorandum apply to vehicles that will be entered in the 1997 American Tour de Sol. However, the specifications were prepared to be general enough for use by other teams and individuals interested in developing hybrid electric vehicles. The purpose of the specifications is to ensure that the vehicles developed do not present a safety hazard to the teams that build and drive them or to the judges, sponsors, or public who attend the competitions. The specifications are by no means the definitive sources of information on constructing hybrid electric vehicles - as electric and hybrid vehicles technologies advance, so will the standards and practices for their construction. In some cases, the new standards and practices will make portions of these specifications obsolete.

  15. Coulomb blockage of hybridization in two-dimensional DNA arrays

    NASA Astrophysics Data System (ADS)

    Vainrub, Arnold; Pettitt, B. Montgomery

    2002-10-01

    Experiments on DNA microarrays have revealed substantial differences in hybridization thermodynamics between DNA free in solution and surface tethered DNA. Here we develop a mean field model of the Coulomb effects in two-dimensional DNA arrays to understand the binding isotherms and thermal denaturation of the double helix. We find that the electrostatic repulsion of the assayed nucleic acid from the array of DNA probes dominates the binding thermodynamics, and thus causes the Coulomb blockage of the hybridization. The results explain, observed in DNA microarrays, the dramatic decrease of the hybridization efficiency and the thermal denaturation curve broadening as the probe surface density grows. We demonstrate application of the theory for evaluation and optimization of the sensitivity, specificity, and the dynamic range of DNA array devices.

  16. Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization.

    PubMed

    Virtanen, M; Syvänen, A C; Oram, J; Söderlund, H; Ranki, M

    1984-12-01

    A cytomegalovirus (CMV)-specific sandwich hybridization test was constructed by using two adjacent BamHI DNA fragments of CMV DNA as reagents. The fragments were cloned into two different vectors. One of the recombinants was attached to the filter, and the other was the labeled probe. When present in the sample, CMV DNA mediated labeling of the filter by hybridizing to both the filter-bound DNA and the probe. The sandwich hybridization test was applied for the detection of CMV DNA from urine. DNA was released from virus by 2% Sarkosyl, concentrated by 2-butanol extraction and isopropanol precipitation, denatured, and finally subjected to the sandwich hybridization test. As a result, 70 to 90% of the original viral DNA could be recovered and demonstrated by the quantitative hybridization reaction. Urine could be stored at room temperature in Sarkosyl for at least 2 days without affecting the detectability of CMV. The clinical applicability of the test was evaluated by studying urine samples from four infants excreting CMV. Sandwich hybridization demonstrated the presence of CMV DNA in all of the specimens. These contained originally 10(5) to 10(8) CMV DNA molecules per ml. PMID:6097598

  17. 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

  18. APPLICATION OF DNA-DNA COLONY HYBRIDIZATION TO THE DETECTION OF CATABOLIC GENOTYPES IN ENVIRONMENTAL SAMPLES

    EPA Science Inventory

    The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFRI and RSF101...

  19. Chromosome-specific DNA Repeat Probes

    SciTech Connect

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

    2006-03-16

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

  20. DNA fluorescence shift sensor: a rapid method for the detection of DNA hybridization using silver nanoclusters.

    PubMed

    Lee, Shin Yong; Hairul Bahara, Nur Hidayah; Choong, Yee Siew; Lim, Theam Soon; Tye, Gee Jun

    2014-11-01

    DNA-templated silver nanoclusters (AgNC) are a class of subnanometer sized fluorophores with good photostability and brightness. It has been applied as a diagnostic tool mainly for deoxyribonucleic acid (DNA) detection. Integration of DNA oligomers to generate AgNCs is interesting as varying DNA sequences can result in different fluorescence spectra. This allows a simple fluorescence shifting effect to occur upon DNA hybridization with the hybridization efficiency being a pronominal factor for successful shifting. The ability to shift the fluorescence spectra as a result of hybridization overcomes the issue of background intensities in most fluorescent based assays. Here we describe an optimized method for the detection of single-stranded and double-stranded synthetic forkhead box P3 (FOXP3) target by hybridization with the DNA fluorescence shift sensor. The system forms a three-way junction by successful hybridization of AgNC, G-rich strand (G-rich) to the target DNA, which generated a shift in fluorescence spectra with a marked increase in fluorescence intensity. The DNA fluorescence shift sensor presents a rapid and specific alternative to conventional DNA detection. PMID:25129336

  1. 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.

  2. Direct detection of DNA conformation in hybridization processes.

    PubMed

    Papadakis, George; Tsortos, Achilleas; Bender, Florian; Ferapontova, Elena E; Gizeli, Electra

    2012-02-21

    DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays. PMID:22248021

  3. DNA hybridization and ligation for directed colloidal assembly

    NASA Astrophysics Data System (ADS)

    Shyr, Margaret

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

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

    PubMed Central

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

    2008-01-01

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

  5. 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

  6. 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

  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. Preparation and characterization of imogolite/DNA hybrid hydrogels.

    PubMed

    Jiravanichanun, Nattha; Yamamoto, Kazuya; Kato, Kenichi; Kim, Jungeun; Horiuchi, Shin; Yah, Weng-On; Otsuka, Hideyuki; Takahara, Atsushi

    2012-01-01

    Imogolite is one of the clay minerals contained in volcanic ash soils. The novel hybrid hydrogels were prepared from imogolite nanofibers and DNA by utilizing strong interaction between the aluminol groups on imogolite surface and phosphate groups of DNA. The hybrid hydrogels of imogolite and DNA were prepared in various feed ratios, and their physicochemical properties and molecular aggregation states were investigated in both dispersion and gel states. The maximum DNA content in the hybrid gels was shown in equivalent molar ratio of imogolite and DNA. The physical properties of the hybrid gels were changed by varying DNA blend ratios. In the dispersion state, the hybrid gels showed a fibrous structure of imogolite, whereas a continuous network structure was observed in pure imogolite, indicating that the hybrid with DNA enhanced the dispersion of imogolite. In the gel state, DNA and imogolite nanofibers formed a 3D network structure. PMID:22148683

  9. Process of labeling specific chromosomes using recombinant repetitive DNA

    DOEpatents

    Moyzis, R.K.; Meyne, J.

    1988-02-12

    Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

  10. 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)

  11. Hybrid DNA materials for energy storage

    NASA Astrophysics Data System (ADS)

    Norwood, R. A.; Thomas, J.; Peyghambarian, N.; Wang, J.; Li, L.; Ouchen, F.; Grote, J. E.

    2010-08-01

    We investigate the dielectric and electrical properties of sol-gel/DNA-CTMA blends, with particular interest in capacitor applications in energy storage. Methacryloyloxypropyltrimethoxysilane (MAPTMS) was the solgel precursor, and DNA-CTMA was blended in to the resulting sol-gel at various weight percentages. The blends were tested for their dielectric properties and dielectric breakdown strength; the 5% DNA blend was found to be optimal with a dielectric constant in the range of 7.5, while the breakdown strength was greater than 800 V/μm for 1 μm films and about 500 V/μm for 5μm films. Hybrid sol-gel/DNA-CTMA/barium titanate nanoparticle composites were also formulated and their dielectric properties measured. While a high dielectric constant was achieved (38), this came at the expense of a significantly reduced breakdown voltage (160V/μm). We discuss these results as well as other aspects of the dielectric and electrical properties of these blends.

  12. 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

  13. Male-specific DNA markers from African catfish (Clarias gariepinus).

    PubMed

    Kovács, B; Egedi, S; Bártfai, R; Orbán, L

    2000-01-01

    We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species. PMID:11766847

  14. DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes.

    PubMed

    Hövelmann, Felix; Seitz, Oliver

    2016-04-19

    The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we

  15. 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. PMID:27317997

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  17. Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

    PubMed Central

    Wang, Jingjing; Morabito, Kenneth; Tang, Jay X.; Tripathi, Anubhav

    2013-01-01

    The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein. PMID:24404041

  18. 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.

  19. 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.

  20. 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.

  1. Applications of Strand-Specific in situ Hybridization

    SciTech Connect

    Goodwin, E.H.; Meyne, J.; Bailey, S.M.; Quigley, D.; Smith, L.; Tennyson, R.

    1997-01-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Fluorescence in situ hybridization (FISH) is used to determine the location of specific DNA sequences on chromosomes. It is an effective tool in genomic mapping and is finding increasing use in medical diagnosis. A ''strand-specific'' version of FISH has been developed in the Life Sciences Division of LANL. The new procedure, named CO-FISH, reveals not only location but also the 5'-to-3'direction of a target sequence, such as the sense strand of a gene. This project was designed to investigate applications of the new technique. Strand-specific FISH was found to be useful and informative for genomic mapping of repetitive DNA sequences. The method provide a valuable new tool for investigating the mechanisms of aneuploidy inducing agents and the cytogenetic phenomena called lateral asymmetry. Finally, using strand-specific FISH, the authors were able to detect certain types of chromosome aberrations (isochromosomes, inversions and Robertsonian translocations) that can be difficult to observe with standard techniques.

  2. Voltammetric detection of sequence-selective DNA hybridization related to Toxoplasma gondii in PCR amplicons.

    PubMed

    Gokce, Gultekin; Erdem, Arzum; Ceylan, Cagdas; Akgöz, Muslum

    2016-03-01

    This work describes the single-use electrochemical DNA biosensor technology developed for voltammetric detection of sequence selective DNA hybridization related to important human and veterinary pathogen; Toxoplasma gondii. In the principle of electrochemical label-free detection assay, the duplex of DNA hybrid formation was detected by measuring guanine oxidation signal occured in the presence of DNA hybridization. The biosensor design consisted of the immobilization of an inosine-modified (guanine-free) probe onto the surface of pencil graphite electrode (PGE), and the detection of the duplex formation in connection with the differential pulse voltammetry(DPV) by measuring the guanine signal. Toxoplasma gondii capture probe was firstly immobilized onto the surface of the activated PGE by wet adsorption. The extent of hybridization at PGE surface between the probe and the target was then determined by measuring the guanine signal observed at +1.0V. The electrochemical monitoring of optimum DNA hybridization has been performed in the target concentration of 40µg/mL in 50min of hybridization time. The specificity of the electrochemical biosensor was then tested using non-complementary, or mismatch short DNA sequences. Under the optimum conditions, the guanine oxidation signal indicating full hybridization was measured in various target concentration from 0.5 to 25µg/mL and a detection limit was found to be 1.78µg/mL. This single-use biosensor platform was successfully applied for the voltammetric detection of DNA hybridization related to Toxoplasma gondii in PCR amplicons. PMID:26717837

  3. Characterization of two DNA probes specific for Serpulina hyodysenteriae.

    PubMed Central

    Sotiropoulos, C; Smith, S C; Coloe, P J

    1993-01-01

    Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology. Images PMID:8349750

  4. Sequence-specific DNA nicking endonucleases.

    PubMed

    Xu, Shuang-yong

    2015-08-01

    A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications. PMID:26352356

  5. 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

  6. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    PubMed

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-01-01

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results. PMID:26345863

  7. 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…

  8. Identification of Lactobacillus UFV H2B20 (probiotic strain) using DNA-DNA hybridization

    PubMed Central

    de Magalhães, J.T.; Uetanabaro, A.P. T.; de Moraes, C.A.

    2008-01-01

    Sequence analyses of the 16S rDNA gene and DNA-DNA hybridization tests were performed for identification of the species of the probiotic Lactobacillus UFV H2b20 strain. Using these two tests, we concluded that this strain, originally considered Lact. acidophilus, should be classified as Lact. delbrueckii. PMID:24031263

  9. Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome.

    PubMed Central

    Clarke, L; Carbon, J

    1975-01-01

    Using a poly(dA-dT) "connector" method, a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle containing one full-length molecule of poly(dT)-tailed DNA from E1 colicinogenic factor (Col E1) fragmented by EcoRI endonuclease annealed to any one of a collection of poly(dA)-tailed linear DNA fragments of the entire E. coli genome. This annealed, but unligated, hybrid DNA was used to transform several different auxotrophic mutants of E. coli, and by direct selection, bacterial clones were isolated which contained specific hybrid plasmids. In this manner, bacterial strains containing Col E1 hybrid plasmids carrying the entire tryptophan operon or the arabinsoe and leucine operons were isolated. The methods described should allow the molecular cloning of any portion of the E. coli genome by selection from a pool of DNA molecules containing at least several hundred different hybrids representing the entire bacterial genome. Images PMID:1105581

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  11. 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.

  12. Comparing Charge Transport in Oligonucleotides: RNA:DNA Hybrids and DNA Duplexes.

    PubMed

    Li, Yuanhui; Artés, Juan M; Qi, Jianqing; Morelan, Ian A; Feldstein, Paul; Anantram, M P; Hihath, Joshua

    2016-05-19

    Understanding the electronic properties of oligonucleotide systems is important for applications in nanotechnology, biology, and sensing systems. Here the charge-transport properties of guanine-rich RNA:DNA hybrids are compared to double-stranded DNA (dsDNA) duplexes with identical sequences. The conductance of the RNA:DNA hybrids is ∼10 times higher than the equivalent dsDNA, and conformational differences are determined to be the primary reason for this difference. The conductance of the RNA:DNA hybrids is also found to decrease more rapidly than dsDNA when the length is increased. Ab initio electronic structure and Green's function-based density of states calculations demonstrate that these differences arise because the energy levels are more spatially distributed in the RNA:DNA hybrid but that the number of accessible hopping sites is smaller. These combination results indicate that a simple hopping model that treats each individual guanine as a hopping site is insufficient to explain both a higher conductance and β value for RNA:DNA hybrids, and larger delocalization lengths must be considered. PMID:27145167

  13. 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

  14. Imaging Specific Genomic DNA in Living Cells.

    PubMed

    Chen, Baohui; Guan, Juan; Huang, Bo

    2016-07-01

    The three-dimensional organization of the genome plays important roles in regulating the functional output of the genome and even in the maintenance of epigenetic inheritance and genome stability. Here, we review and compare a number of newly developed methods-especially those that utilize the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system-that enable the direct visualization of specific, endogenous DNA sequences in living cells. We also discuss the practical considerations in implementing the CRISPR imaging technique to achieve sufficient signal-to-background levels, high specificity, and high labeling efficiency. These DNA labeling methods enable tracking of the copy number, localization, and movement of genomic elements, and we discuss the potential applications of these methods in understanding the searching and targeting mechanism of the Cas9-sgRNA complex, investigating chromosome organization, and visualizing genome instability and rearrangement. PMID:27145877

  15. Optical fibre-based detection of DNA hybridization.

    PubMed

    Hine, Anna V; Chen, Xianfeng; Hughes, Marcus D; Zhou, Kaiming; Davies, Edward; Sugden, Kate; Bennion, Ian; Zhang, Lin

    2009-04-01

    A dual-peak LPFG (long-period fibre grating), inscribed in an optical fibre, has been employed to sense DNA hybridization in real time, over a 1 h period. One strand of the DNA was immobilized on the fibre, while the other was free in solution. After hybridization, the fibre was stripped and repeated detection of hybridization was achieved, so demonstrating reusability of the device. Neither strand of DNA was fluorescently or otherwise labelled. The present paper will provide an overview of our early-stage experimental data and methodology, examine the potential of fibre gratings for use as biosensors to monitor both nucleic acid and other biomolecular interactions and then give a summary of the theory and fabrication of fibre gratings from a biological standpoint. Finally, the potential of improving signal strength and possible future directions of fibre grating biosensors will be addressed. PMID:19290879

  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. A novel fluorescent reagent for recognition of triplex DNA with high specificity and selectivity.

    PubMed

    Chen, Zongbao; Zhang, Huimi; Ma, Xiaoming; Lin, Zhenyu; Zhang, Lan; Chen, Guonan

    2015-11-21

    A fluorescent agent (DMT) was screened for recognizing triplex DNA with a specific and selective characteristic, which was embedded into the triplex DNA structure. The triplex DNA was firstly formed by a complementary target sequence through two distinct and sequential events. The conditions including pH and hybridization time, fluorescent agent concentration and embedding time were optimized in the experiment. Under the optimum conditions, the fluorescence signal was enhanced up to 9-fold in comparison with the DMT embedding into the ssDNA, dsDNA and G-quadruplexes. Under the same fluorescence conditions, the changes of the fluorescence signal were also investigated by several kinds of base mismatched DNAs in the experiment. The results showed that our biosensor provided excellent discrimination efficiency toward the perfectly mismatched target DNA with no formation of triplex DNA. We preliminarily deduced the mechanism of the fluorescent reagent for recognizing triplex DNA with high specificity and selectivity. PMID:26456316

  18. Scanning Tunneling Microscopy of DNA-Carbon Nanotube Hybrids

    NASA Astrophysics Data System (ADS)

    Yarotski, Dzmitry; Kilina, Svetlana; Talin, Alec; Balatsky, Alexander; Tretiak, Sergei; Taylor, Antoinette

    2009-03-01

    Production of carbon nanotube-based (CNT) devices holds a great promise for bringing the size of electronic circuits down to molecular scales. Recently, yet another step has been made towards achieving this goal by developing a new method for metal-semiconductor CNT separation, which relies on wrapping the CNT with ssDNA molecule[1]. Though it was shown that the outcome of the separation process strongly depends on the DNA sequence, further investigations have to be conducted to determine detailed structure of the hybrids and their electronic properties. Here, we use STM to characterize structural and electronic properties of the CNT-DNA hybrids and compare experimental results to theoretical calculations. STM images reveal 3.3 nm DNA coiling period, which agrees very well with the theoretical predictions. Additional width modulations with characteristic lengths of 1.9 and 2.6 nm are observed along the molecule itself. Although scanning tunneling microscopy confirms the presence of DNA in the hybrid and visualizes its structure, further experimental work is required to reveal the dependence of electronic properties of hybrids on their internal structure. [1] M. Zheng et al., Science 302, 1545 (2004).

  19. 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.

  20. Post-hybridization recovery of membrane filter-bound DNA for enzymatic DNA amplification.

    PubMed

    Chong, K Y; Chen, C M; Choo, K B

    1993-04-01

    We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes. PMID:8476600

  1. 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

  2. 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

  3. 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

  4. Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

    SciTech Connect

    Arlinghaus, H.F.; Kwoka, M.N.; Guo, X.Q.; Jacobson, K.B.

    1997-04-15

    A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched tin isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. 34 refs., 5 figs.

  5. Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection.

    PubMed

    Peterson, Eric M; Manhart, Michael W; Harris, Joel M

    2016-06-21

    Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability

  6. 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.

  7. DNA-DNA hybridization assay for detection of Salmonella spp. in foods.

    PubMed Central

    Fitts, R; Diamond, M; Hamilton, C; Neri, M

    1983-01-01

    We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology. Images PMID:6360046

  8. 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 0.4fM. Furthermore, the biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences. PMID:26872213

  9. 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. PMID:25177907

  10. DNA microarrays for hybridization detection by surface plasmon resonance spectroscopy.

    PubMed

    Kick, Alfred; Bönsch, Martin; Katzschner, Beate; Voigt, Jan; Herr, Alexander; Brabetz, Werner; Jung, Martin; Sonntag, Frank; Klotzbach, Udo; Danz, Norbert; Howitz, Steffen; Mertig, Michael

    2010-12-15

    We report on the development of a new platform technology for the detection of genetic variations by means of surface plasmon resonance (SPR) spectroscopy. TOPAS chips with integrated optics were exploited in combination with microfluidics. Within minutes, the detection of hybridization kinetics was achieved simultaneously at all spots of the DNA microarray. A nanoliter dispenser is used to deposit thiol-modified single-stranded probe DNA on the gold surface of the chips. We investigated the influence of different parameters on hybridization using model polymerase chain reaction (PCR) products. These PCR products comprised a single-stranded tag sequence being complementary to an anti-tag sequence of probes immobilized on the gold surface. The signals increased with increasing length of PCR products (60, 100 or 300 base pairs) as well as with their concentration. We investigated hybridizations on DNA microarrays comprising 90 spots of probe DNA with three different sequences. Furthermore, we demonstrate that sequences with possible hairpin structures significantly lower the binding rate, and thus, the SPR signals during hybridization. PMID:20729067

  11. Co-Localization of Multiple Antigens and Specific DNA

    PubMed Central

    Mueller, Marcus; Wacker, Karin; Hickey, William F.; Ringelstein, Erich B.; Kiefer, Reinhard

    2000-01-01

    Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies. PMID:11106556

  12. DNA sequence copy number analysis by Comparative Genomic Hybridization (CGH)

    SciTech Connect

    Pinkel, D.; Kallioniemi, A.; Kallioniemi, O.; Waldman, F.; Sudar, D.; Gray, I. ); Rutovitz, D.; Piper, I. )

    1993-01-01

    Comparative Genomic Hybridization (CGH) uses the kinetics of in situ hybridization to compare the copy numbers of different DNA sequences within the same genome and the copy numbers of the same sequences among different genomes. In a typical application genomic DNA from a tumor and from normal cells are differentially labeled and simultaneously hybridized to normal metaphase chromosomes, and detected with different fluorochromes. Properly registered images of each fluorochrome are obtained using a microscope equipped with multi-band filters and a CCD camera. Digital image analysis permits measurement of intensity ratio profiles along each of the target chromosomes. Studies of cells with known aberrations indicate that the intensity ratio at each position is proportional to the ratio of the copy numbers of the sequences that bind there in the tumor and normal genomes. Analytical challenges posed by the need to efficiently obtain copy number karyotypes are discussed.

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

    PubMed Central

    2013-01-01

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

  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. Rapid genomic DNA changes in allotetraploid fish hybrids.

    PubMed

    Wang, J; Ye, L H; Liu, Q Z; Peng, L Y; Liu, W; Yi, X G; Wang, Y D; Xiao, J; Xu, K; Hu, F Z; Ren, L; Tao, M; Zhang, C; Liu, Y; Hong, Y H; Liu, S J

    2015-06-01

    Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals. PMID:25669608

  17. 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.

  18. Multilayer DNA origami packed on hexagonal and hybrid lattices.

    PubMed

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

    2012-01-25

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

  19. Physical localisation of repetitive DNA sequences in Alstroemeria: karyotyping of two species with species-specific and ribosomal DNA.

    PubMed

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

    1997-10-01

    Fluorescence in situ hybridization (FISH) was used to localise two species-specific repetitive DNA sequences, A001-I and D32-13, and two highly conserved 25S and 5S rDNA sequences on the metaphase chromosomes of two species of Alstroemeria. The Chilean species, Alstroemeria aurea (2n = 16), has abundant constitutive heterochromatin, whereas the Brazilian species, Alstroemeria inodora, has hardly any heterochromatin. The A. aurea specific A001-I probe hybridized specifically to the C-band regions on all chromosomes. The FISH patterns on A. inodora chromosomes using species-specific probe D32-13 resembled the C-banding pattern and the A001-I pattern on A. aurea chromosomes. There were notable differences in number and distribution of rDNA sites between the two species. The 25S rDNA probe revealed 16 sites in A. aurea that closely colocalised with A001-I sites and 12 in A. inodora that were predominantly detected in the centromeric regions. FISH karyotypes of the two Alstroemeria species were constructed accordingly, enabling full identification of all individual chromosomes. These FISH karyotypes will be useful for monitoring the chromosomes of both Alstroemeria species in hybrids and backcross derivatives. PMID:9352644

  20. 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. PMID:25286352

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

    PubMed

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

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  2. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

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

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  3. ATP-Responsive DNA-Graphene Hybrid Nanoaggregates for Anticancer Drug Delivery

    PubMed Central

    Mo, Ran; Jiang, Tianyue; Sun, Wujin; Gu, Zhen

    2015-01-01

    Stimuli-triggered drug delivery systems are primarily focused on the applications of the tumor microenvironmental or cellular physiological cues to enhance the release of drugs at the target site. In this study, we applied adenosine-5′-triphosphate (ATP), the primary “energy molecule”, as a trigger for enhanced release of preloaded drugs responding to the intracellular ATP concentration that is significantly higher than the extracellular level. A new ATP-responsive anticancer drug delivery strategy utilizing DNA-graphene crosslinked hybrid nanoaggregates as carriers was developed for controlled release of doxorubicin (DOX), which consists of graphene oxide (GO), two single-stranded DNA (ssDNA, denoted as DNA1 and DNA2) and ATP aptamer. The single-stranded DNA1 and DNA2 together with the ATP aptamer serve as the linkers upon hybridization for controlled assembly of the DNA-GO nanoaggregates, which effectively inhibited the release of DOX from the GO nanosheets. In the presence of ATP, the responsive formation of the ATP/ATP aptamer complex causes the dissociation of the aggregates, which promoted the release of DOX in the environment with a high ATP concentration such as cytosol compared with that in the ATP-deficient extracellular fluid. This supports the development of a novel ATP-responsive platform for targeted on-demand delivery of anticancer drugs inside specific cells. PMID:25736497

  4. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  5. 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

  6. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    PubMed Central

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  7. Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

    PubMed Central

    Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

    1986-01-01

    Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

  8. 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.

  9. 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.

  10. Using radiation hybrids to generate region-specific markers for human chromosome 9

    SciTech Connect

    Britt, D.E.; Mark, H.F.L.; Nebres, M.

    1994-09-01

    The production of sequence tagged sites and polymorphic markers is an important step in generating a physical map of the human genome and identifying loci involved in genetic diseases. Our work involves the physical mapping of the short arm of human chromosome 9, the site of at least one tumor supressor gene, as well as the locus involved in cartilage hair hypoplasia. Our goal is to increase the number of markers available for 9p, using a panel of radiation hybrids we have constructed and characterized. The hybrids were generated from a monochromosomal hybrid that contains human chromosome 9 marked with a retroviral vector. Radiation hybrids were produced that contain overlapping regions of the chromosome surrounding the site of retroviral integration. In order to generate markers specific for the short arm, Alu-PCR products from a radiation hybrid containing only 9p were cloned. Clones were mapped back to a subpanel of hybrids and grouped into intervals. By using a hybrid subpanel containing overlapping portions 9p, we are able to identify clones from defined regions. DNA from the clones was sequenced and this information used to generate sequence tagged sites. We have also developed a number of new polymorphic markers, taking advantage of the high degree of polymorphism of the 3{prime} end of each Alu sequence. For each polymorphic marker, a specific primer was designed from the cloned DNA and then paired with an Alu primer. These primer pairs were used to amplify DNA from unrelated individuals, in order to identify primer sets that detect useful polymorphisms. Both the STS and polymorphic markers will be extremely useful in the construction of a physical map of chromosome 9, and in the identification of genes on the short arm of the chromosome.

  11. Quantum dot monolayer for surface plasmon resonance signal enhancement and DNA hybridization detection.

    PubMed

    Ghrera, Aditya Sharma; Pandey, Manoj Kumar; Malhotra, Bansi Dhar

    2016-06-15

    We report results of studies relating to the fabrication of a surface plasmon resonance (SPR)-based nucleic acid sensor for quantification of DNA sequence specific to chronic myelogeneous leukemia (CML). The SPR disk surface has been modified with octadecanethiol self-assembled monolayer followed by deposition of the tri-n-octylphosphine oxide capped cadmium selenide quantum dots (QD) Langmuir monolayer. The deposition is performed via Langmuir-Blodgett (LB) technique. For the sensor chip preparation, covalent immobilization of the thiol-terminated DNA probe sequence (pDNA) using displacement reaction is accomplished. This integrated SPR chip has been used to detect target complementary DNA concentration by monitoring the change in coupling angle via hybridization. It is revealed that this biosensor exhibits high sensitivity (0.7859 m(0)pM(-1)) towards complementary DNA and can be used to detect it in the concentration range, 180 pM to 5 pM with detection limit as 4.21 pM. The results of kinetic studies yield the values of hybridization and dissociation rate constants as 9.6 × 10(4) M(-1) s(-1) and 2.3 × 10(-2) s(-1), respectively, with the equilibrium constant for hybridization as 4.2 × 10(6) M(-1). PMID:26878485

  12. Probing DNA hybridization efficiency and single base mismatch by X-ray photoelectron spectroscopy.

    PubMed

    Liu, Zheng-Chun; Zhang, Xin; He, Nong-Yue; Lu, Zu-Hong; Chen, Zhen-Cheng

    2009-07-01

    We demonstrated the use of X-ray photoelectron spectroscopy (XPS) to study DNA hybridization. Target DNA labeled with hexachloro-fluorescein (HEX) was hybridized to DNA arrays with four different probes. Each probe dot of the hybridized arrays was detected with XPS. The XPS Cl2p peak areas were found to decrease with an increase in mismatched bases in DNA probes. The Cl2p core-level peak area ratio of a probe perfectly matched to one, two and three base-mismatched probes accorded well with the results of conventional fluorescent imaging, which shows that XPS is a potential tool for analyzing DNA arrays. The DNA arrays' hybridization efficiency was assessed by the molar ratio of chlorine to phosphorus in a DNA strand, which was determined from the relevant XPS Cl2p and P2p core-level peak areas after hybridization. This could provide a new method to detect DNA hybridization efficiency. PMID:19282155

  13. Antibody specific for a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-07-11

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  14. Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

    PubMed

    Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. PMID:23968861

  15. 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

  16. 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.

  17. Preparation of genosensor for detection of specific DNA sequence of the hepatitis B virus

    NASA Astrophysics Data System (ADS)

    Honorato Castro, Ana C.; França, Erick G.; de Paula, Lucas F.; Soares, Marcia M. C. N.; Goulart, Luiz R.; Madurro, João M.; Brito-Madurro, Ana G.

    2014-09-01

    An electrochemical genosensor was constructed for detection of specific DNA sequence of the hepatitis B virus, based on graphite electrodes modified with poly(4-aminophenol) and incorporating a specific oligonucleotide probe. The modified electrode containing the probe was evaluated by differential pulse voltammetry, before and after incubation with the complementary oligonucleotide target. Detection was performed by monitoring oxidizable DNA bases (direct detection) or using ethidium bromide as indicator of the hybridization process (indirect detection). The device showed a detection limit for the oligonucleotide target of 2.61 nmol L-1. Indirect detection using ethidium bromide was promising in discriminating mismatches, which is a very desirable attribute for detection of disease-related point mutations. In addition, it was possible to observe differences between hybridized and non-hybridized surfaces by atomic force microscopy.

  18. 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

  19. Molecular characterization of a bovine Y-specific DNA sequence conserved in taurine and zebu breeds.

    PubMed

    Alves, Beatriz C A; Mayer, Mário G; Taber, Anna Paula; Egito, Andréa A; Fagundes, Valéria; McElreavey, Ken; Moreira-Filho, Carlos A

    2006-06-01

    The identification of new bovine male-specific DNA sequences is of great interest because the bovine Y chromosome remains poorly characterized in terms of physical and genetic maps. Since taurine and zebu Y chromosomes are structurally different, the identification of Y-specific sequences present in both sub-species is particularly important: these sequences are of evolutionary significance and can be broadly used for embryo sexing. In this work, we initially used the random amplified polymorphic DNA (RAPD) technique to search for male-specific sequences present as monomorphic markers in genomic DNA from zebu and taurine bulls. A male-specific RAPD band was found to be present and highly conserved in both sub-species, as demonstrated by Southern blotting, fluorescent in situ hybridization (FISH) and DNA sequencing. In a previous work, a pair of primers derived from this marker was successfully used in taurine and zebu embryo sexing. PMID:17286047

  20. DNA methylation in placentas of interspecies mouse hybrids.

    PubMed Central

    Schütt, Sabine; Florl, Andrea R; Shi, Wei; Hemberger, Myriam; Orth, Annie; Otto, Sabine; Schulz, Wolfgang A; Fundele, Reinald H

    2003-01-01

    Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids. PMID:14504229

  1. Energy storage specification requirements for hybrid-electric vehicle

    SciTech Connect

    Burke, A.F.

    1993-09-01

    A study has been made of energy storage unit requirements for hybrid-electric vehicles. The drivelines for these vehicles included both primary energy storage units and/or pulse power units. The primary energy storage units were sized to provide ``primary energy`` ranges up to 60 km. The total power capability of the drivelines were such that the vehicles had 0 to 100 km/h acceleration times of 10 to 12 s. The power density requirements for primary energy storage devices to be used in hybrid vehicles are much higher than that for devices to be used in electric vehicles. The energy density and power density requirements for pulse-power devices for hybrid vehicles, are not much different than those in an electric vehicle. The cycle life requirements for primary energy-storage units for hybrid vehicles are about double that for electric vehicles, because of the reduced size of the storage units in the hybrid vehicles. The cycle life for pulse-power devices for hybrid vehicles is about the same as for electric vehicles having battery load leveling. Because of the need for additional components in the hybrid driveline, the cost of the energy storage units in hybrid vehicles should be much less (at least a factor of two) than those in electric vehicles. There are no presently available energy storage units that meet all the specifications for hybrid vehicle applications, but ultracapacitors and bipolar lead-acid batteries are under development that have the potential for meeting them. If flywheel systems having a mechanical system energy density of 40 to 50 W{center_dot}h/kg and an electrical system power density of 2 to 3 kw/kg can be developed, they would have the potential of meeting specifications for primary storage and pulse power units.

  2. 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.

  3. 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

  4. Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

    PubMed Central

    de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

    1988-01-01

    It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment. PMID:2453027

  5. POU proteins bend DNA via the POU-specific domain.

    PubMed Central

    Verrijzer, C P; van Oosterhout, J A; van Weperen, W W; van der Vliet, P C

    1991-01-01

    POU proteins constitute a family of ubiquitous as well as cell type-specific transcription factors that share the conserved POU DNA binding domain. This domain consists of two distinct subdomains, a POU-specific domain and a POU homeodomain, that are both required for high affinity sequence-specific DNA binding. In a circular permutation assay, several POU proteins, including Oct-1, Oct-2A, Oct-6 and Pit-1, demonstrated a position dependent mobility of the protein-DNA complexes, suggesting induction of DNA bending. This was confirmed by detection of relative bend direction, using pre-bent DNA, and by enhanced ligase mediated cyclization. Bending was caused by interaction with the POU domain. By contrast, binding of the POU homeodomain did not distort the DNA structure, indicating that the POU-specific domain confers DNA bending. Images PMID:1915275

  6. 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.

  7. [Use of photo-anchoring of DNA probes for fluorescent in situ hybridization].

    PubMed

    Nasedkina, T V; Mal'kov, R B; Fedorova, L I; Godovikova, T S; Kolpashchikov, D M; Poletaev, A I

    1998-01-01

    A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization. PMID:9821246

  8. Sequence-Specific DNA Binding by a Short Peptide Dimer

    NASA Astrophysics Data System (ADS)

    Talanian, Robert V.; McKnight, C. James; Kim, Peter S.

    1990-08-01

    A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4^circC. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

  9. 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.

  10. Cloning of a short HLA-DQ beta locus-specific cDNA probe: typing for DQw specificities.

    PubMed

    Sood, S K; McCusker, C T; Singal, D P

    1989-01-01

    A short HLA-DQ beta locus-specific (141 bp) probe was cloned from the full-length pII-beta-1 cDNA. Pst 1-digested genomic DNA from homozygous typing cell lines (HTC) was hybridized with this short DQ beta locus-specific, pDQ beta 141, probe. Restriction fragment length polymorphism (RFLP) patterns generated with this DQ beta locus-specific probe were compared with those obtained with the full-length (627 bp) DQ beta, pII-beta-1, probe. The results demonstrate that the RFLP patterns with the pDQ beta 141 probe were very simple, and no crossreacting DR beta and DX beta bands were observed. DQw1, 2, 3 and 4 specificities could each be identified by a single RFLP. PMID:2467193

  11. Denaturation, renaturation, and loss of DNA during in situ hybridization procedures.

    PubMed

    Raap, A K; Marijnen, J G; Vrolijk, J; van der Ploeg, M

    1986-05-01

    With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes. PMID:3709305

  12. 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. PMID:25290445

  13. Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.

    PubMed Central

    Rodtong, S; Dobbinson, S; Thode-Andersen, S; McConnell, M A; Tannock, G W

    1993-01-01

    Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples. Images PMID:8285690

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

    PubMed Central

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

    2014-01-01

    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 colour 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 10 pM, which is about 100-fold lower than that of traditional unamplified homogeneous assays. PMID:25500528

  15. 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

  16. The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA.

    PubMed

    Wang, Jing; McCord, Bruce

    2011-06-01

    A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples. PMID:21706494

  17. 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

  18. 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.

  19. 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

  20. [Determination of fetal sex by amplification of Y-chromosome-specific DNA].

    PubMed

    Huang, S Z

    1989-08-01

    This paper describes a rapid and highly sensitive method for determination of fetal sex. Y-chromosome-specific sequences as well as Alu-specific sequences were amplified with polymerase chain reaction (PCR). Then fetal sex was determined by comparison of the two amplified DNA sequences. Polymerase chain reaction can be performed on lysed amniotic fluid cells or chorionic villus samples or dried blood spots on filter paper blot without prior DNA extraction. The analysis of the amplified DNA was performed immediately by agarose gel electrophoresis without DNA hybridization with radioactive probe. By using this method, determination of sex was performed on 3 fetuses at risk for DMD, and on 2 fetuses at risk for hemophilia, prenatal detection was confirmed by examination of the neonates. PMID:2620273

  1. Synthetic Nucleotides as Probes of DNA Polymerase Specificity

    PubMed Central

    Walsh, Jason M.; Beuning, Penny J.

    2012-01-01

    The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides. PMID:22720133

  2. 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

  3. Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

    PubMed

    Leach, T J; Glaser, R L

    1998-10-15

    Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect. PMID:9753752

  4. Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

    PubMed Central

    Géron-Landre, Bénédicte; Roulon, Thibaut; Desbiolles, Pierre; Escudé, Christophe

    2003-01-01

    Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules. PMID:14530458

  5. DNA hybridization assay for detection of nucleopolyhedrovirus in whitemarked tussock moth (Orgyia leucostigma) larvae.

    PubMed

    Ebling, P M; Smith, P A; van Frankenhuyzen, K

    2001-01-01

    DNA dot-blot hybridization assays utilizing a horseradish peroxidase-labelled whole genomic DNA probe and enhanced chemiluminescence were conducted to quantify detection thresholds of nucleopolyhedrovirus (NPV) in whitemarked tussock moth (Orgyia leucostigma) larvae. The minimum detection thresholds for an aqueous suspension of occlusion bodies (OBs), OBs added to macerates of non-infected larvae and OBs in macerates of diseased larvae were 7.8 x 10(3), 7.8 x 10(3), and 1.5 x 10(3) OBs, respectively. Purified viral DNA was detected at a concentration of 1.6 x 10(-1) ng in a 20 microliters volume. The presence of pre-occluded viral nucleocapsids and DNA, inherent to infected larvae, improved the detection threshold five-fold compared with OBs alone. Larval tissues did not block the detection system utilized, nor did they bind non-specifically to the probe. Detection thresholds, upon sequential hybridization of the same membrane, on average deteriorated two-fold between the first and second hybridization and an additional six-fold between the second and third hybridization. NPV infection was detected two days post-inoculation (pi) in about one-third of the larvae examined and in almost all larvae three days pi. Microscopic analysis of stained larval smears missed NPV infection in almost all larvae two days pi and about two-thirds of the larvae three days pi. Results from the two methods of analysis were not comparable until four days pi. The detection system utilized is a reliable, efficient and simple method for the early detection of NPV infection in large numbers of larvae and may be used for further studies quantifying the role of this baculovirus in the ecology of whitemarked tussock moth populations. PMID:11455634

  6. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

    SciTech Connect

    D'Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. ); Antonacci, R. )

    1993-11-01

    The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

  7. 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.

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

    PubMed Central

    Orth, Gérard; Jeanteur, Philippe; Croissant, Odile

    1971-01-01

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

  9. Identification of Brugia malayi in vectors with a species-specific DNA probe.

    PubMed

    Sim, B K; Mak, J W; Cheong, W H; Sutanto, I; Kurniawan, L; Marwoto, H A; Franke, E; Campell, J R; Wirth, D F; Piessens, W F

    1986-05-01

    We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis. PMID:3518507

  10. Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum.

    PubMed

    León, G; Martinez, M A; Etchegaray, J P; Vera, M I; Figueroa, J; Krauskopf, M

    1994-03-01

    To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova. PMID:24420936

  11. Control of Rate-Bounded Hybrid Systems with Liveness Specifications

    NASA Technical Reports Server (NTRS)

    Heymann, Michael; Lin, Feng; Meyer, George

    1998-01-01

    In the present paper we examine the control problem for a class of composite hybrid machines (CHMs) that consist of concurrent operation (employing synchronous composition) of elementary hybrid machines (EHMs), that allows both signal sharing and event synchronization. A controller can then be coupled with the plant by means of synchronous composition. We confine our attention to controllers that interact with the system only through event synchronization. We present an initial investigation of synthesis of liveness controllers for hybrid machines. To this end we define open hybrid machines as systems that can interact with the environment through event synchronization and can be therefor be "driven" to their marked configuration by user (controller). Liveness specifications must be associated with timing constraints. We may require that for a specified time limit, every run reach a marked configuration within that time limit. Alternatively, a more relaxed specification may be that, for some (unspecified) global time bound, every run of the system reach a marked configuration within that time bound. Finally, the least restrictive liveness requirement is that every run reach a marked configuration within a finite time limit (but we do not insist on the existence of a global time bound for all runs).

  12. SELECTION OF INTERSPECIFIC SUGARCANE HYBRIDS USING MICROSATELLITE DNA MARKERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three types of species-specific DNA markers, namely, PCR, RAPD, and microsatellites, have been recently developed at the USDA-ARS, SRRC, Sugarcane Research Unit, Houma, Louisiana. Of these, the microsatellite markers are the most polymorphic and can produce distinctive fingerprints (or molecular al...

  13. 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

  14. 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.

  15. A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

    PubMed Central

    Matheson, V G; Munakata-Marr, J; Hopkins, G D; McCarty, P L; Tiedje, J M; Forney, L J

    1997-01-01

    A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates. PMID:9212434

  16. 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.

  17. DNA linking number change induced by sequence-specific DNA-binding proteins

    PubMed Central

    Chen, Bo; Xiao, Yazhong; Liu, Chang; Li, Chenzhong; Leng, Fenfei

    2010-01-01

    Sequence-specific DNA-binding proteins play a key role in many fundamental biological processes, such as transcription, DNA replication and recombination. Very often, these DNA-binding proteins introduce structural changes to the target DNA-binding sites including DNA bending, twisting or untwisting and wrapping, which in many cases induce a linking number change (ΔLk) to the DNA-binding site. Due to the lack of a feasible approach, ΔLk induced by sequence-specific DNA-binding proteins has not been fully explored. In this paper we successfully constructed a series of DNA plasmids that carry many tandem copies of a DNA-binding site for one sequence-specific DNA-binding protein, such as λ O, LacI, GalR, CRP and AraC. In this case, the protein-induced ΔLk was greatly amplified and can be measured experimentally. Indeed, not only were we able to simultaneously determine the protein-induced ΔLk and the DNA-binding constant for λ O and GalR, but also we demonstrated that the protein-induced ΔLk is an intrinsic property for these sequence-specific DNA-binding proteins. Our results also showed that protein-mediated DNA looping by AraC and LacI can induce a ΔLk to the plasmid DNA templates. Furthermore, we demonstrated that the protein-induced ΔLk does not correlate with the protein-induced DNA bending by the DNA-binding proteins. PMID:20185570

  18. Highly specific electronic signal transduction mediated by DNA/metal self-assembly.

    SciTech Connect

    Dentinger, Paul M.; Pathak, Srikant

    2003-11-01

    Highly specific interactions between DNA could potentially be amplified if the DNA interactions were utilized to assemble large scale parts. Fluidic assembly of microsystem parts has the potential for rapid and accurate placement of otherwise difficult to handle pieces. Ideally, each part would have a different chemical interaction that allowed it to interact with the substrate only in specific areas. One easy way to obtain a multiple chemical permutations is to use synthetic DNA oligomers. Si parts were prepared using silicon-on-insulator technology microfabrication techniques. Several surface chemistry protocols were developed to react commercial oligonucleotides to the parts. However, no obvious assembly was achieved. It was thought that small defects on the surface did not allow the microparts to be in close enough proximity for DNA hybridization, and this was. in part, confirmed by interferometry. To assist in the hybridization, plastic, pliable parts were manufactured and a new chemistry was developed. However, assembly was still absent even with the application of force. It is presently thought that one of three mechanisms is preventing the assembly. The surfaces of the two solid substrates can not get in close enough proximity, the surface chemistry lacks sufficient density to keep the parts from separating, or DNA interactions in close proximity on solid substrates are forbidden. These possibilities are discussed in detail.

  19. Specific suppression of anti-DNA production in vitro

    SciTech Connect

    Liebling, M.R.; Wong, C.; Radosevich, J.; Louie, J.S.

    1988-09-01

    To investigate the regulation of anti-DNA antibody production, we generated anti-DNA-specific suppressor cells by exposing normal human T cells and a small percentage of adherent cells to high concentrations of DNA. These cells suppressed the production of anti-DNA by both autologous peripheral blood mononuclear cells (PBMC) and allogeneic PBMC derived from systemic lupus erythematosus (SLE) patients. Anti-DNA production was suppressed significantly more than anti-RNA, antitetanus, or total immunoglobulin production. Specific suppression was enhanced by increasing the numbers of DNA-primed CD8+ cells and was obliterated by irradiation of the DNA-primed cells. In contrast to T cells from normal individuals, T cells obtained from two intensively studied SLE patients were unable to generate specific suppressor cells for anti-DNA production in both autologous and allogeneic test systems. Despite this defect, these patients were still capable of generating specific suppressor cells for antibody production directed against an exogenous antigen, tetanus toxoid.

  20. Nonlinear dynamics of specific DNA-protein interactions

    NASA Astrophysics Data System (ADS)

    Dwiputra, D.; Hidayat, W.; Khairani, R.; Zen, F. P.

    2016-03-01

    Interactions between DNA binding protein and specific base pairs of nucleic acid is critical for biological process. We propose a new model of DNA-protein interactions to depict the dynamics of specific DNA-protein interactions. Hydrogen bonds (H-bonds) are, among the other intermolecular interactions in DNA, the most distinctive in term of specificity of molecular bonds. As H-bonds account for specificity, we only consider the dynamics affected by H-bonds between DNA base pairs and H-bonds connecting protein side chains and DNA. The H-bonds are modelled by Morse potentials and coupling terms in the Hamiltonian of coupled oscillators resembling a coupling between planar DNA chain and a protein molecule. In this paper we give a perturbative approach as an attempt for a soliton solution. The solution is in the form of nonlinear travelling wave having the amplitudes satisfying coupled nonlinear Schrodinger equations and is interpreted as the mediator for nonlocal transmittance of biological information in DNA.

  1. Global and gene specific DNA methylation changes during zebrafish development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  2. DNA-melamine hybrid molecules: from self-assembly to nanostructures.

    PubMed

    Kumari, Rina; Banerjee, Shib Shankar; Bhowmick, Anil K; Das, Prolay

    2015-01-01

    Single-stranded DNA-melamine hybrid molecular building blocks were synthesized using a phosphoramidation cross-coupling reaction with a zero linker approach. The self-assembly of the DNA-organic hybrid molecules was achieved by DNA hybridization. Following self-assembly, two distinct types of nanostructures in the form of linear chains and network arrays were observed. The morphology of the self-assembled nanostructures was found to depend on the number of DNA strands that were attached to a single melamine molecule. PMID:26199847

  3. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-10-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. PMID:8548693

  4. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed Central

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-01-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. Images Fig. 2. Fig. 3. PMID:8548693

  5. DNA-sequence-specific erasers of epigenetic memory.

    PubMed

    Mozgova, Iva; Köhler, Claudia

    2016-05-27

    How epigenetic regulators find their specific targets remains a challenging question. Two parallel studies show that REF6, a plant H3K27me3 demethylase, binds a specific DNA motif via its zinc-finger domains and recruits the SWI/SNF-type ATPase BRAHMA, demonstrating a sequence-specific recruitment mechanism for a chromatin-modifying complex. PMID:27230685

  6. 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

  7. 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

  8. 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

  9. RNA∶DNA hybrids initiate quasi-palindrome-associated mutations in highly transcribed yeast DNA.

    PubMed

    Kim, Nayun; Cho, Jang-Eun; Li, Yue C; Jinks-Robertson, Sue

    2013-11-01

    RNase H enzymes promote genetic stability by degrading aberrant RNA:DNA hybrids and by removing ribonucleotide monophosphates (rNMPs) that are present in duplex DNA. Here, we report that loss of RNase H2 in yeast is associated with mutations that extend identity between the arms of imperfect inverted repeats (quasi-palindromes or QPs), a mutation type generally attributed to a template switch during DNA synthesis. QP events were detected using frameshift-reversion assays and were only observed under conditions of high transcription. In striking contrast to transcription-associated short deletions that also are detected by these assays, QP events do not require Top1 activity. QP mutation rates are strongly affected by the direction of DNA replication and, in contrast to their elevation in the absence of RNase H2, are reduced when RNase H1 is additionally eliminated. Finally, transcription-associated QP events are limited by components of the nucleotide excision repair pathway and are promoted by translesion synthesis DNA polymerases. We suggest that QP mutations reflect either a transcription-associated perturbation of Okazaki-fragment processing, or the use of a nascent transcript to resume replication following a transcription-replication conflict. PMID:24244191

  10. Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.

    PubMed

    Orum, H; Nielsen, P E; Jørgensen, M; Larsson, C; Stanley, C; Koch, T

    1995-09-01

    Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure. PMID:7495562

  11. 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

  12. An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

    PubMed

    Chen, Sherry Xi; Seelig, Georg

    2016-04-20

    Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology. PMID:27010123

  13. 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.

  14. Molecular cytogenetics of Alstroemeria: identification of parental genomes in interspecific hybrids and characterization of repetitive DNA families in constitutive heterochromatin.

    PubMed

    Kuipers, A G; van Os, D P; de Jong, J H; Ramanna, M S

    1997-02-01

    The genus Alstroemeria consists of diploid (2n = 2x = 16) species originating mainly from Chile and Brazil. Most cultivars are triploid or tetraploid interspecific hybrids. C-banding of eight species revealed obvious differentiation of constitutive heterochromatin within the genus. The present study focused on the molecular (cyto)genetic background of this differentiation. Genomic slot-blot analysis demonstrated strong conservation of major parts of the genomes among six species. The chromosomes of A. aurea and A. ligtu, species with pronounced interstitial C-bands, were found to contain large amounts of highly repetitive and species-specific DNA. The variation in size, number and intensity of strongly probed bands of major repetitive DNA families observed in genomic Southern blots of Sau3A, HaeIII, and MseI digests indicated a strong correlation between variation in genomic DNA composition and different C-banding patterns among Alstroemeria species. Genomic in situ hybridization (GISH) revealed a clear distinction between parental chromosomes in the hybrids between Chilean and Brazilian species and also between Chilean species, as long as at least one of the parental species possessed prominent C-banding. Regarding the latter, discriminative hybridization resulted from highly repetitive species specific DNA in the heterochromatic chromosome regions of A. aurea and A. ligtu, and caused GISH banding patterns that coincided with the C-banding patterns. PMID:9088641

  15. Spatially localized generation of nucleotide sequence-specific DNA damage.

    PubMed

    Oh, D H; King, B A; Boxer, S G; Hanawalt, P C

    2001-09-25

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues. PMID:11572980

  16. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution

    SciTech Connect

    Kreader, C.A.

    1995-04-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans. Although the B. vulgatus probes detected high levels of their target DNA in most of the house pets, similarly high levels of target DNA were found only in a few individuals from other groups of nonhumans. Therefore, the results indicate that these probes can distinguish human from non human feces in many cases. 50 refs., 5 figs., 2 tabs.

  17. Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

    PubMed Central

    Jones, D H; Winistorfer, S C

    1992-01-01

    We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information. Images PMID:1371352

  18. Colorimetric detection of DNA hybridization based on a dual platform of gold nanoparticles and graphene oxide.

    PubMed

    Thavanathan, Jeevan; Huang, Nay Ming; Thong, Kwai Lin

    2014-05-15

    The unique property of gold nanoparticles (Au NP) to induce colour change and the versatility of graphene oxides (GO) in surface modification makes them ideal in the application of colorimetric biosensor. Thus we developed a label free optical method to detect DNA hybridization through a visually observed colour change. The Au NP is conjugated to a DNA probe and is allowed to hybridize with the DNA target to the GO thus causing a change in colour from pinkish-red to purplish blue. Spectrophometry analysis gave a wavelength shift of 22 nm with 1 µM of DNA target. Sensitivity testing using serially diluted DNA conjugated GO showed that the optimum detection was at 63 nM of DNA target with the limit at 8 nM. This proves the possibility for the detection of DNA hybridization through the use of dual nanoparticle system by visual observation. PMID:24368225

  19. Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna

    NASA Astrophysics Data System (ADS)

    Hamad-Schifferli, Kimberly; Schwartz, John J.; Santos, Aaron T.; Zhang, Shuguang; Jacobson, Joseph M.

    2002-01-01

    Increasingly detailed structural and dynamic studies are highlighting the precision with which biomolecules execute often complex tasks at the molecular scale. The efficiency and versatility of these processes have inspired many attempts to mimic or harness them. To date, biomolecules have been used to perform computational operations and actuation, to construct artificial transcriptional loops that behave like simple circuit elements and to direct the assembly of nanocrystals. Further development of these approaches requires new tools for the physical and chemical manipulation of biological systems. Biomolecular activity has been triggered optically through the use of chromophores, but direct electronic control over biomolecular `machinery' in a specific and fully reversible manner has not yet been achieved. Here we demonstrate remote electronic control over the hybridization behaviour of DNA molecules, by inductive coupling of a radio-frequency magnetic field to a metal nanocrystal covalently linked to DNA. Inductive coupling to the nanocrystal increases the local temperature of the bound DNA, thereby inducing denaturation while leaving surrounding molecules relatively unaffected. Moreover, because dissolved biomolecules dissipate heat in less than 50picoseconds (ref. 16), the switching is fully reversible. Inductive heating of macroscopic samples is widely used, but the present approach should allow extension of this concept to the control of hybridization and thus of a broad range of biological functions on the molecular scale.

  20. Transcriptome screen for fast evolving genes by Inter-Specific Selective Hybridization (ISSH)

    PubMed Central

    2010-01-01

    Background Fast evolving genes are targets of an increasing panel of biological studies, from cancer research to population genetics and species specific adaptations. Yet, their identification and isolation are still laborious, particularly for non-model organisms. We developed a method, named the Inter-Specific Selective Hybridization (ISSH) method, for generating cDNA libraries enriched in fast evolving genes. It utilizes transcripts of homologous tissues of distinct yet related species. Experimental hybridization conditions are monitored in order to discard transcripts that do not find their homologous counterparts in the two species sets as well as transcripts that display a strong complementarity between the two species. Only heteroduplexes that disanneal at low stringency are used for constructing the resulting cDNA library. Results We demonstrate the efficiency of the ISSH method by generating a brain cDNA library enriched in fast evolving transcripts of a non-model catfish species as well as a control, non-enriched library. Our results indicate that the enriched library contains effectively more fast evolving sequences than the control library. Gene annotation analyses also indicate enrichment in genes with low expression levels and non-ubiquitously expressed genes, both categories encompassing the majority of fast evolving genes. Furthermore, most of the identified transcripts show higher sequence divergence between two closely related catfish species as compared to recognized fast evolving DNA markers. Conclusions The ISSH method offers a simple, inexpensive and efficient way to screen the transcriptome for isolating fast evolving genes. This method opens new opportunities in the investigation of biological mechanisms that include fast evolving genes, such as the evolution of lineage specific processes and traits responsible for species adaptation to their environment. PMID:20175901

  1. 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

  2. The minimal amount of starting DNA for Agilent's hybrid capture-based targeted massively parallel sequencing.

    PubMed

    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

  3. Activation of different split functionalities on re-association of RNA-DNA hybrids.

    PubMed

    Afonin, Kirill A; Viard, Mathias; Martins, Angelica N; Lockett, Stephen J; Maciag, Anna E; Freed, Eric O; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A

    2013-04-01

    Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications. PMID:23542902

  4. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

    PubMed Central

    Cortés-Gutiérrez, Elva I.; Ortíz-Hernández, Brenda L.; Dávila-Rodríguez, Martha I.; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

    2013-01-01

    We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1. PMID:23429197

  5. Quantitative modeling of transcription factor binding specificities using DNA shape.

    PubMed

    Zhou, Tianyin; Shen, Ning; Yang, Lin; Abe, Namiko; Horton, John; Mann, Richard S; Bussemaker, Harmen J; Gordân, Raluca; Rohs, Remo

    2015-04-14

    DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function. PMID:25775564

  6. 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.

  7. The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome.

    PubMed

    Oikawa, Haruna; Tun, Zaw; Young, David R; Ozawa, Hiroyasu; Yamazaki, Kentaro; Tanaka, Einosuke; Honda, Katsuya

    2002-09-20

    Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself. PMID:12237124

  8. Three dimensional dual labelled DNA fluorescent in situ hybridization analysis in fixed tissue sections

    PubMed Central

    Kernohan, Kristin D.; Bérubé, Nathalie G.

    2014-01-01

    Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections. PMID:26150931

  9. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  11. T-Specific DNA Polymorphisms among Wild Mice from Israel and Spain

    PubMed Central

    Figueroa, F.; Neufeld, E.; Ritte, U.; Klein, J.

    1988-01-01

    Lehrach and his coworkers have isolated a series of DNA probes that specifically hybridize with different regions of mouse chromosome 17 within the t complex. The probes display restriction fragment length polymorphisms, RFLPs, which are specific for the t haplotypes in all laboratory mouse strains tested thus far. Some of these probes have been used to test wild mice populations for these t-associated DNA forms. It is demonstrated that populations from Germany, Switzerland, Italy, Greece, Yugoslavia, Australia, Costa Rica, and Venezuela contain chromosomes in which all the tested DNA loci display the t-specific polymorphisms. The frequency of mice carrying these chromosomes is as high as 31%. Wild mice from Israel and Spain, on the other hand, carry chromosomes displaying t-specific DNA forms only at one or two of the probed loci, while the other loci carry the wild-type (+) forms. These chromosomes thus resemble the partial t haplotypes known from the study of laboratory mice. One possible interpretation of these findings is that these DNA polymorphisms contributed to the assembly of the complete t haplotypes and that these haplotypes may have originated in the Middle East. PMID:3165081

  12. Spatially localized generation of nucleotide sequence-specific DNA damage

    PubMed Central

    Oh, Dennis H.; King, Brett A.; Boxer, Steven G.; Hanawalt, Philip C.

    2001-01-01

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen–DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320–400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA–psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen–TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues. PMID:11572980

  13. Blocking oligo--a novel approach for improving chip-based DNA hybridization efficiency.

    PubMed

    Tao, Sheng-ce; Gao, Hua-fang; Cao, Fei; Ma, Xue-mei; Cheng, Jing

    2003-08-01

    For most of the commonly used DNA chips, the probes are usually single-stranded oligonucleotides and the targets are double-stranded DNAs (dsDNAs). Only one strand of the DNA serves as the target while the other competes with the probes immobilized on the chip for the target and therefore is regarded as the interfering strand. In this report, a novel technique was developed for improving the hybridization efficiency on DNA chips by using blocking oligos, which is complimentary to the target interfering strand to reduce the influence of the interfering strand. The hybridization efficiency of dsDNA was much lower than that of single-stranded DNA (ssDNA) when synthesized DNA targets were tested on the DNA chip. Blocking oligos can improve the hybridization efficiency of dsDNA to about 2/3 that of ssDNA. Blocking oligos have also been applied to PCR products of different lengths for hybridization. The hybridization efficiency with blocking oligos is about three times higher than that without blocking oligos. We have tested PCR products of 1054 and 435 bp using our blocking procedure, and the results are consistent. PMID:12944123

  14. Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II).

    PubMed Central

    Dreyer, G B; Dervan, P B

    1985-01-01

    The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA X Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions. Images PMID:3919391

  15. 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.

  16. DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe.

    PubMed

    Maruyama, Tatsuo; Park, Lian-Chun; Shinohara, Toshimitsu; Goto, Masahiro

    2004-01-01

    We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm. PMID:14715007

  17. Characterization of Vibrio metschnikovii and Vibrio gazogenes by DNA-DNA hybridization and phenotype.

    PubMed Central

    Farmer, J J; Hickman-Brenner, F W; Fanning, G R; Gordon, C M; Brenner, D J

    1988-01-01

    Vibrio metschnikovii and Vibrio gazogenes are two new Vibrio species that have been little studied. Thirteen strains of V. metschnikovii were highly related to the type strain, NCTC 8443, by DNA-DNA hybridization. Relatedness values were 83 to 90% at 60 degrees C and 75 to 84% at the more stringent 75 degrees C. Divergence values ranged from 0.7 to 1.9. Strains of V. metschnikovii were oxidase negative and did not reduce nitrate to nitrite. The other phenotypic characteristics agreed with published data. Twenty-three strains of V. gazogenes were isolated from salt marshes and marshy areas on the coast of North and South Carolina. A new medium, marine agar supplemented with an additional 2.5% agar, reduced the problem of swarming by marine Vibrio species and enhanced the isolation of V. gazogenes and other organisms. By DNA-DNA hybridization, 22 of 23 strains were 76% or more related to the type strain of V. gazogenes, ATCC 29988. However, four DNA hybridization subgroups were defined on the basis of divergence values and/or phenotype. Strains of DNA group 1 were more highly related to each other, and this group contained the type strain and six other strains. Strains of DNA group 2 were more highly related to each other, and this group contained reference strain ATCC 43942 and 14 other strains. Strains of DNA group 1 did not ferment melibiose or D-sorbitol (one strain was sorbitol positive), but strains of DNA group 2 fermented both sugars. A revised phenotypic description of V. gazogenes based on 24 strains was written on the basis of reactions (within 2 days of incubation) at 25 degrees C in media supplemented with Na+, K+, and Mg2+. Positive results (100% positive unless indicated) included motility; gas production during fermentation (96% at 2 days, 100% at 3 to 7 days); growth in nutrient broth with the addition of 1% NaCl (88%), 2% NaCl, 3.5% NaCl, 6% NaCl, 8% NaCl, and 10% NaCl (92%); dry red or orange colonies on marine agar; and fermentation of L

  18. 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.

  19. DNA Markers Associated with General and Specific Cognitive Abilities.

    ERIC Educational Resources Information Center

    Petrill, Stephen A.; And Others

    1996-01-01

    Data on specific cognitive abilities for 86 children ages 6 to 12 from an allelic association study found three DNA markers significantly associated with specific cognitive ability scales after the effects of general intelligence were removed. These preliminary results support the hierarchical model predicted by genetic research. (SLD)

  20. Advances in the theory and practice of DNA-hybridization as a systematic method.

    PubMed

    Sheldon, F H

    1994-01-01

    DNA hybridization continues in the 1990s to provide insight into phylogeny and evolution. The resilience of this 30-year-old distance technique may be attributed to its fundamental power as a comparative method, as well as to advances in our understanding of its operation and improvements in experimental design and data analysis. These attributes and advances, along with the assumptions and limitations of DNA hybridization, are discussed in this paper. Examples are provided of recent DNA hybridization studies of molecular, morphological, and behavioral systematics and evolution. PMID:7994110

  1. Self-assembly and hybridization mechanisms of DNA with cationic polythiophene.

    PubMed

    Rubio-Magnieto, Jenifer; Azene, Elias Gebremedhn; Knoops, Jérémie; Knippenberg, Stefan; Delcourt, Cécile; Thomas, Amandine; Richeter, Sébastien; Mehdi, Ahmad; Dubois, Philippe; Lazzaroni, Roberto; Beljonne, David; Clément, Sébastien; Surin, Mathieu

    2015-08-28

    The combination of DNA and π-conjugated polyelectrolytes (CPEs) represents a promising approach to develop DNA hybridization biosensors, with potential applications for instance in the detection of DNA lesions and single-nucleotide polymorphisms. Here we exploit the remarkable optical properties of a cationic poly[3-(6'-(trimethylphosphonium)hexyl)thiophene-2,5-diyl] (CPT) to decipher the self-assembly of DNA and CPT. The ssDNA/CPT complexes have chiroptical signatures in the CPT absorption region that are strongly dependent on the DNA sequence, which we relate to differences in supramolecular interactions between the thiophene monomers and the various nucleobases. By studying DNA-DNA hybridization and melting processes on preformed ssDNA/CPT complexes, we observe sequence-dependent mechanisms that can yield DNA-condensed aggregates. Heating-cooling cycles show that non-equilibrium mixtures can form, noticeably depending on the working sequence of the hybridization experiment. These results are of high importance for the use of π-conjugated polyelectrolytes in DNA hybridization biosensors and in polyplexes. PMID:26179509

  2. A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates.

    PubMed Central

    Li, C; Ferguson, D A; Ha, T; Chi, D S; Thomas, E

    1993-01-01

    HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies. Images PMID:8370744

  3. Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy

    PubMed Central

    Dutta, Samrat; Armitage, Bruce A.; Lyubchenko, Yuri L.

    2016-01-01

    Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplex. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. (2011) Journal of Organic Chemistry 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γMPPNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that γMPPNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 0.030 ± 0.01 sec-1 for γMPPNA-DNA hybrid duplex vs. 0.375 ± 0.18 sec-1 for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γMPPNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γMPPNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes. PMID:26898903

  4. Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

    PubMed

    Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

    2016-03-15

    Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes. PMID:26898903

  5. A search for specificity in DNA-drug interactions.

    PubMed

    Cruciani, G; Goodford, P J

    1994-06-01

    The GRID force field and a principal component analysis have been used in order to predict the interactions of small chemical groups with all 64 different triplet sequences of B-DNA. Factors that favor binding to guanine-cytosine base pairs have been identified, and a dictionary of ligand groups and their locations is presented as a guide to the design of specific DNA ligands. PMID:7918250

  6. DNA-directed mutations. Leading and lagging strand specificity

    NASA Technical Reports Server (NTRS)

    Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

    1999-01-01

    The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

  7. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    PubMed Central

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-01-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5′-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer. PMID:24135881

  8. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    NASA Astrophysics Data System (ADS)

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-10-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.

  9. 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. PMID:25457775

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

    PubMed

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

    2016-02-14

    Here we show the formation of the complex between a DNA aptamer and a single-walled carbon nanotube (SWNT) and its reaction with its target protein. The aptamer, which is specifically bound with thrombin, the target protein in this study, easily wraps and disperses the SWNT by noncovalent π-π stacking. PMID:26763942

  11. 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. PMID:25128622

  12. Specificity of damage recognition and catalysis of DNA repair.

    PubMed

    Osman, R; Fuxreiter, M; Luo, N

    2000-05-01

    A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton

  13. Measuring thermodynamic details of DNA hybridization using fluorescence

    PubMed Central

    You, Yong; Tataurov, Andrey V; Owczarzy, Richard

    2011-01-01

    Modern real-time PCR systems make it easy to monitor fluorescence while temperature is varied for hundreds of samples in parallel, permitting high-throughput studies. We employed such system to investigate melting transitions of ordered nucleic acid structures into disordered random coils. Fluorescent dye and quencher were attached to oligonucleotides in such a way that changes of fluorescence intensity with temperature indicated progression of denaturation. When fluorescence melting data were compared with traditional ultraviolet optical experiments, commonly used dye/quencher combinations, like fluorescein and tetramethylrhodamine, showed substantial discrepancies. We have therefore screened 22 commercially available fluorophores and quenchers for their ability to reliably report annealing and melting transitions. Dependence of fluorescence on temperature and pH was also investigated. The optimal performance was observed using Texas Red or ROX dyes with Iowa Black RQ or Black Hole quenchers. These labels did not alter two-state nature of duplex melting process and provided accurate melting temperatures, free energies, enthalpies, and entropies. We also suggest a new strategy for determination of DNA duplex thermodynamics where concentration of a dye-labeled strand is kept constant and its complementary strand modified with a quencher is added at increasing excess. These methodological improvements will help build predictive models of nucleic acid hybridization. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 472–486, 2011. PMID:21384337

  14. DNA-based hybridization chain reaction for an ultrasensitive cancer marker EBNA-1 electrochemical immunosensor.

    PubMed

    Song, Chao; Xie, Guoming; Wang, Li; Liu, Lingzhi; Tian, Guang; Xiang, Hua

    2014-08-15

    An ultrasensitive and selective electrochemical immunosensor was developed for the detection of Epstein Barr virus nuclear antigen 1 (EBNA-1). Firstly, a suspension of graphene sheets (GS) and multi-walled carbon nanotubes (MWCNTs) was prepared with the aid of chitosan (CS) solution and then modified on a glassy carbon electrode (GCE). Gold nanoparticles (AuNPs) were then electrodeposited onto the surface of the GS-MWCNTs film by cyclic voltammetry (CV) to immobilize the captured antibodies. After that, specific sandwich immunoreactions were formed among the captured antibody, EBNA-1, and secondary antibody, DNA-coated carboxyl multi-wall carbon nanotubes (DNA-MWCNTs-Ab2). DNA initiator strands (S0) and secondary antibodies linked to the MWCNTs and double-helix DNA polymers were obtained by hybridization chain reaction (HCR), and here S0 on the MWCNTs propagates a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Finally, electroactive indicator doxorubicin hydrochloride was intercalated into the CG-GC steps between the HCR products and could produce an electrochemical signal, which was monitored by differential pulse voltammetry (DPV). Under optimum conditions, the amperometric signal increased linearly with the target concentrations (0.05-6.4ngmL(-1)), and the immunosensor exhibited a detection limit as low as 0.7pgmL(-1) (S/N=3). The proposed method showed acceptable stability and reproducibility, as well as favorable recovery for EBNA-1 in human serum. The proposed immunosensor provides a novel avenue for signal amplification and potential applications in bioanalysis and clinical diagnostics. PMID:24632131

  15. Electronic hybridization detection in microarray format and DNA genotyping

    PubMed Central

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-01-01

    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. PMID:24569823

  16. 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.

  17. Tissue-specific patterns of allelically-skewed DNA methylation

    PubMed Central

    Marzi, Sarah J.; Meaburn, Emma L.; Dempster, Emma L.; Lunnon, Katie; Paya-Cano, Jose L.; Smith, Rebecca G.; Volta, Manuela; Troakes, Claire; Schalkwyk, Leonard C.; Mill, Jonathan

    2016-01-01

    ABSTRACT While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ∼220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood. PMID:26786711

  18. Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

    PubMed

    Smyth, J A; Weston, J; Moffett, D A; Todd, D

    2001-11-01

    Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent. PMID:11724137

  19. 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

  20. Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi.

    PubMed

    Desquesnes, Marc; Marc, Desquesnes; Kamyingkird, Ketsarin; Ketsarin, Kamyingkird; Yangtara, Sarawut; Sarawut, Yangtara; Milocco, Cristina; Cristina, Milocco; Ravel, Sophie; Sophie, Ravel; Wang, Ming-Hui; Ming-Hui, Wang; Lun, Zhao-Rong; Zhao-Rong, Lun; Morand, Serge; Serge, Morand; Jittapalapong, Sathaporn; Sathaporn, Jittapalapong

    2011-08-01

    Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1-10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes. PMID:21570489

  1. Specifications of an accelerator for the soliton hybrid reactor (RHYS)

    NASA Astrophysics Data System (ADS)

    Gaveau, Bernard; Maillard, Jacques; Maurel, Gérard; Silva, Jorge

    2006-06-01

    The soliton hybrid reactor is a concept of an Accelerator Driven System, with a design insuring a long lifetime without core interventions. Soliton reactors and "candle reactors" have been proposed in order to use reactors for very long periods without reprocessing or enrichment. In this paper, we present the concept of hybrid soliton reactor. In this system, the constant displacement of the beam during the 30 years lifetime implies lower constraints on the window compared to other accelerator-driven systems. During its lifetime, the reactor can present constant profiles in chemical and isotopic composition and in power production if the beam of protons is maintained within certain limits. This is the soliton-like behavior. Using a mathematical analysis of the problem, we have shown that the solution of the equations presents a solitary wave behavior which is stable if the accelerator intensity is sufficiently low or if the velocity of the neutron source is sufficiently high. We have simulated these two behaviors with a GEANT III Monte Carlo program: a soliton behavior, and a one which may become unstable when the intensity is too large or the velocity too small. These preliminary considerations allow us to describe some specifications concerning an accelerator which can drive such a system.

  2. 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

  3. pH-dependent specific binding and combing of DNA.

    PubMed Central

    Allemand, J F; Bensimon, D; Jullien, L; Bensimon, A; Croquette, V

    1997-01-01

    Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 PMID:9336201

  4. Comparative study of antipneumocystis agents in rats by using a Pneumocystis carinii-specific DNA probe to quantitate infection.

    PubMed Central

    Liberator, P A; Anderson, J W; Powles, M; Pittarelli, L A; Worley, M; Becker-Hapak, M; Graves, D C; Schmatz, D M

    1992-01-01

    A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods. Images PMID:1452667

  5. DNA topology confers sequence specificity to nonspecific architectural proteins.

    PubMed

    Wei, Juan; Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

    2014-11-25

    Topological constraints placed on short fragments of DNA change the disorder found in chain molecules randomly decorated by nonspecific, architectural proteins into tightly organized 3D structures. The bacterial heat-unstable (HU) protein builds up, counter to expectations, in greater quantities and at particular sites along simulated DNA minicircles and loops. Moreover, the placement of HU along loops with the "wild-type" spacing found in the Escherichia coli lactose (lac) and galactose (gal) operons precludes access to key recognition elements on DNA. The HU protein introduces a unique spatial pathway in the DNA upon closure. The many ways in which the protein induces nearly the same closed circular configuration point to the statistical advantage of its nonspecificity. The rotational settings imposed on DNA by the repressor proteins, by contrast, introduce sequential specificity in HU placement, with the nonspecific protein accumulating at particular loci on the constrained duplex. Thus, an architectural protein with no discernible DNA sequence-recognizing features becomes site-specific and potentially assumes a functional role upon loop formation. The locations of HU on the closed DNA reflect long-range mechanical correlations. The protein responds to DNA shape and deformability—the stiff, naturally straight double-helical structure—rather than to the unique features of the constituent base pairs. The structures of the simulated loops suggest that HU architecture, like nucleosomal architecture, which modulates the ability of regulatory proteins to recognize their binding sites in the context of chromatin, may influence repressor-operator interactions in the context of the bacterial nucleoid. PMID:25385626

  6. 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.

  7. Short thio-multi-walled carbon nanotubes and Au nanoparticles enhanced electrochemical DNA biosensor for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Guo, Feng; Zhang, Jimei; Dai, Zhao; Zheng, Guo

    2010-07-01

    A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a thio group (MWNTs-SH) and gold nanoparticles (GNPs) for covalent DNA immobilization and enhanced hybridization detection is described. The key step for developing this novel DNA biosensor is to cut the pristine MWNT into short and generate lots of active sites simultaneously. With this approach, the target DNA could be quantified in a linear range from 8.5×10-10 to 1.5×10-5 mol/L, with a detection limit of 1.67×10-11 mol/L by 3σ.

  8. Electrochemical DNA sensor for specific detection of picomolar Hg(II) based on exonuclease III-assisted recycling signal amplification.

    PubMed

    Gan, Xiaorong; Zhao, Huimin; Chen, Shuo; Quan, Xie

    2015-03-21

    An ultrasensitive methodology was successfully developed for the quantitative detection of picomolar Hg(2+) based on the combination of thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and exonuclease III-aided recycling signal amplification. Single-strand probe DNA was immobilized on an Au electrode via an Au-S bond. In the presence of Hg(2+), the probe DNA hybridized with the target DNA containing four thymine-thymine (T-T) mismatches via the Hg(2+)-mediated coordination of T-Hg(2+)-T base pairs. Then the probe DNA in the DNA duplex was specifically recognized and selectively digested by exonuclease III; in contrast the target DNA was safely dissociated from the DNA duplexes to subsequently hybridize with a new signal probe, leading to target recycling and signal amplification. As a result, the peak current caused by the electrostatic interactions of [Ru(NH3)6](3+) cations with the backbone of the probe DNA decreased by different degrees, corresponding to the Hg(2+) concentrations. Under the optimum conditions, the proposed electrochemical DNA biosensor showed a robust detection limit as low as 1 pM (S/N = 3), with a wide linear range from 0.01 to 500 nM and good selectivity. In addition, the proposed method was successfully applied to assay Hg(2+) in real environmental samples. PMID:25676090

  9. Gold coated ferric oxide nanoparticles based disposable magnetic genosensors for the detection of DNA hybridization processes.

    PubMed

    Loaiza, Óscar A; Jubete, Elena; Ochoteco, Estibalitz; Cabañero, German; Grande, Hans; Rodríguez, Javier

    2011-01-15

    In this article, a disposable magnetic DNA sensor using an enzymatic amplification strategy for the detection of specific hybridization processes, based on the coupling of streptavidin-peroxidase to biotinylated target sequences, has been developed. A thiolated 19-mer capture probe was attached to gold coated ferric oxide nanoparticles and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxide was attached to the biotinylated target and the resulting modified gold coated ferric oxide nanoparticles were captured by a magnetic field on the surface of a home-made carbon screen printed electrode (SPE). Using hydroquinone as a mediator, a square wave voltammetric procedure was chosen to detect the hybridization process after the addition of hydrogen peroxide. Different aspects concerning the assay protocol and nanoparticles fabrication were optimized in order to improve the sensitivity of the developed methodology. A low detection limit (31 pM) with good stability (RSD=7.04%, n=10) was obtained without the need of polymerase chain reaction (PCR) amplification. PMID:20951565

  10. Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization.

    PubMed

    Karaballi, R A; Nel, A; Krishnan, S; Blackburn, J; Brosseau, C L

    2015-09-01

    Rapid detection of disease biomarkers at the patient point-of-care is essential to timely and effective treatment. The research described herein focuses on the development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) DNA aptasensor capable of direct detection of tuberculosis (TB) DNA. Specifically, a plausible DNA biomarker present in TB patient urine was chosen as the model target for detection. Cost-effective screen printed electrodes (SPEs) modified with silver nanoparticles (AgNP) were used as the aptasensor platform, onto which the aptamer specific for the target DNA was immobilized. Direct detection of the target DNA was demonstrated through the appearance of SERS peaks characteristic for adenine, present only in the target strand. Modulation of the applied potential allowed for a sizeable increase in the observed SERS response and the use of thiol back-filling prevented non-specific adsorption of non-target DNA. To our knowledge, this work represents the first EC-SERS study of an aptasensor for the direct, label-free detection of DNA hybridization. Such a technology paves the way for rapid detection of disease biomarkers at the patient point-of-care. PMID:25780805

  11. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution.

    PubMed Central

    Kreader, C A

    1995-01-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7538270

  12. Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

    PubMed

    Yeo, Jung Eun; Wickramaratne, Susith; Khatwani, Santoshkumar; Wang, Yen-Chih; Vervacke, Jeffrey; Distefano, Mark D; Tretyakova, Natalia Y

    2014-08-15

    DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  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. PMID:24965286

  14. 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. PMID:26879193

  15. A novel sandwich hybridization method for selecting cDNAs from large genomic regions: Identification of cDNAs from the cloned genomic DNA spanning the XLRP locus

    SciTech Connect

    Yan, D.; McHenry, C.; Fujita, R.

    1994-09-01

    We have developed an efficient hybridization-based cDNA-selection method. A sandwich of three species - single-stranded cDNA, tagged RNA derived from genomic DNA, and biotinylated RNA complementary to the tag - allows specific retention of hybrids on an avidin-matrix. Previously, using model experiments, we demonstrated highly specific and efficient selection of a retinal gene, NRL, from complex mixtures of cDNA clones, using a sub-library from a 5 kb NRL genomic clone. We have now applied this selection strategy to isolate cDNAs from human adult retina and fetal eye libraries, with the {open_quotes}genomic RNA{close_quotes} derived from two YAC clones (OTC-C and 55B) spanning the region of X-linked retinitis pigmentosa (XLRP) locus RP3 at Xp21.1. Effectiveness of the selection-method was monitored by enrichment of TCTEX-1L gene that maps within the 55B YAC. Of the 15 selected cDNA clones that hybridized to the 55B YAC DNA, five appear to the map to specific cosmid clones derived from the 55B YAC. Inserts in these selected cDNA clones range from 0.5 to 2.3 kb in size. Additional clones are now being isolated and characterized. This procedure should be independent of the size or complexity of genomic DNA being used for selection, allow for the isolation of full-length cDNAs, and may have wider application.

  16. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  17. Hybridization and Introgression among Species of Sunfish (Lepomis): Analysis by Mitochondrial DNA and Allozyme Markers

    PubMed Central

    Avise, John C.; Saunders, Nancy C.

    1984-01-01

    We explore the potential of mitochondrial DNA (mtDNA) analysis, alone and in conjunction with allozymes, to study low-frequency hybridization and introgression phenomena in natural populations. MtDNAs from small samples of nine species of sunfish (Lepomis, Centrarchidae) were purified and digested with each of 13 informative restriction enzymes. Digestion profiles for all species were highly distinct: estimates of overall fragment homology between pairs of species ranged from 0–36%. Allozymes encoded by nine nuclear genes also showed large frequency differences among species and together with mtDNA provided many genetic markers for hybrid identification. A genetic analysis of 277 sunfish from two locations in north Georgia revealed the following: (1) a low frequency of interspecific hybrids, all of which appeared to be F1's; (2) the involvement of five sympatric Lepomis species in the production of these hybrids; (3) no evidence for introgression between species in our study locales (although for rare hybridization, most later-generation backcrosses would not be reliably distinguished from parentals); (4) a tendency for hybridizations to take place preferentially between parental species differing greatly in abundance; (5) a tendency for the rare species in a hybrid cross to provide the female parent. Our data suggest that absence of conspecific pairing partners and mating stimuli for females of rarer species may be important factors in increasing the likelihood of interspecific hybridization. The maternal inheritance of mtDNA offers at least two novel advantages for hybridization analysis: (1) an opportunity to determine direction in hybrid crosses; and (2) due to the linkage among mtDNA markers, an increased potential to distinguish effects of introgression from symplesiomorphy or character convergence. PMID:6090268

  18. 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

  19. Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

    PubMed Central

    Hwang, Michael T.; Landon, Preston B.; Lee, Joon; Choi, Duyoung; Mo, Alexander H.; Glinsky, Gennadi; Lal, Ratnesh

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine. PMID:27298347

  20. Highly specific SNP detection using 2D graphene electronics and DNA strand displacement.

    PubMed

    Hwang, Michael T; Landon, Preston B; Lee, Joon; Choi, Duyoung; Mo, Alexander H; Glinsky, Gennadi; Lal, Ratnesh

    2016-06-28

    Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine. PMID:27298347

  1. Site-Specific Surface Functionalization of Gold Nanorods Using DNA Origami Clamps.

    PubMed

    Shen, Chenqi; Lan, Xiang; Lu, Xuxing; Meyer, Travis A; Ni, Weihai; Ke, Yonggang; Wang, Qiangbin

    2016-02-17

    Precise control over surface functionalities of nanomaterials offers great opportunities for fabricating complex functional nanoarchitectures but still remains challenging. In this work, we successfully developed a novel strategy to modify a gold nanorod (AuNR) with specific surface recognition sites using a DNA origami clamp. AuNRs were encapsulated by the DNA origami through hybridization of single-stranded DNA on the AuNRs and complementary capture strands inside the clamp. Another set of capture strands on the outside of the clamp create the specific recognition sites on the AuNR surface. By means of this strategy, AuNRs were site-specifically modified with gold nanoparticles at the top, middle, and bottom of the surface, respectively, to construct a series of well-defined heterostructures with controlled "chemical valence". Our study greatly expands the utility of DNA origami as a tool for building complex nanoarchitectures and represents a new approach for precise tailoring of nanomaterial surfaces. PMID:26824749

  2. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

  3. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  4. Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

    PubMed Central

    Bullwinkle, Tammy J.; Samorodnitsky, Daniel; Rosati, Rayna C.; Koudelka, Gerald B.

    2012-01-01

    We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein. PMID:22509323

  5. A DNA hybridization detection based on fluorescence resonance energy transfer between dye-doped core-shell silica nanoparticles and gold nanoparticles.

    PubMed

    Gao, Feng; Cui, Peng; Chen, Xiaoxiao; Ye, Qingqing; Li, Maoguo; Wang, Lun

    2011-10-01

    A novel and efficient method to evaluate the DNA hybridization based on a fluorescence resonance energy transfer (FRET) system, with fluorescein isothiocyanate (FITC)-doped fluorescent silica nanoparticles (SiNPs) as donor and gold nanoparticles (AuNPs) as acceptor, has been reported. The strategy for specific DNA sequence detecting is based on DNA hybridization event, which is detected via excitation of SiNPs-oligonucleotide conjugates and energy transfer to AuNPs-oligonucleotide conjugates. The proximity required for FRET arises when the SiNPs-oligonucleotide conjugates hybridize with partly complementary AuNPs-oligonucleotide conjugates, resulting in the fluorescence quenching of donors, SiNPs-oligonucleotide conjugates, and the formation of a weakly fluorescent complex, SiNPs-dsDNA-AuNPs. Upon the addition of the target DNA sequence to SiNPs-dsDNA-AuNPs complex, the fluorescence restores (turn-on). Based on the restored fluorescence, a homogeneous assay for the target DNA is proposed. Our results have shown that the linear range for target DNA detection is 0-35.0 nM with a detection limit (3σ) of 3.0 picomole. Compared with FITC-dsDNA-AuNPs probe system, the sensitivity of the proposed probe system for target DNA detection is increased by a factor of 3.4-fold. PMID:21845282

  6. 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)

  7. DNA hybrid dielectric film devices for energy storage and bioelectronics applications

    NASA Astrophysics Data System (ADS)

    Joyce, Donna M.; Venkat, Narayanan; Ouchen, Fahima; Singh, Kristi M.; Smith, Steven R.; Grote, James G.

    2013-10-01

    DNA biopolymer hybrids have been investigated for energy storage applications and also as potential high k gate dielectrics in bioelectronics applications such as BioFETs. DNA-based hybrid films incorporating sol-gel-derived ceramics have shown strong promise as insulating dielectrics for high voltage capacitor applications. Our studies of DNA-CTMA complex/sol-gel hybrid thin film devices have demonstrated reproducibility and stability in temperature-and frequency-dependent dielectric properties as well as reliability in DC voltage breakdown measurements, attaining values consistently in the 300 - 350 V/um range. We have also investigated DNA-inorganic hybrids by ex situ blending of aqueous solutions of DNA with high k ceramics such as BaTiO3 and TiO2. These systems are currently being investigated as potential gate dielectrics for BioFETs by virtue of their relatively high dielectric constant, high DC electrical resistivity, and lower leakage currents than pristine DNA. Functionally layered devices have also been designed, fabricated and characterized to determine any added benefit in dielectric applications. The electrical/dielectric characteristics of DNA and DNA-CTMA with sol-gel-derived ceramics, high k ceramic fillers, and in layered devices were examined to determine their effect on vital dielectric parameters for energy storage and bioelectronics applications.

  8. A controlled microfluidic electrochemical lab-on-a-chip for label-free diffusion-restricted DNA hybridization analysis.

    PubMed

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

    2015-02-15

    Lab-on-a-chip (LOC) devices for electrochemical analysis of DNA hybridization events offer a technology for real-time and label-free assessment of biomarkers at the point-of-care. Here, we present a microfluidic LOC, with 3 × 3 arrayed electrochemical sensors for the analysis of DNA hybridization events. A new dual layer microfluidic valved manipulation system is integrated providing controlled and automated capabilities for high throughput analysis. This feature improves the repeatability, accuracy, and overall sensing performance (Fig. 1). The electrochemical activity of the fabricated microfluidic device is validated and demonstrated repeatable and reversible Nernstian characteristics. System design required detailed analysis of energy storage and dissipation as our sensing modeling involves diffusion-related electrochemical impedance spectroscopy. The effect of DNA hybridization on the calculated charge transfer resistance and the diffusional resistance components is evaluated. We demonstrate a specific device with an average cross-reactivity value of 27.5%. The device yields semilogarithmic dose response and enables a theoretical detection limit of 1 nM of complementary ssDNA target. This limit is lower than our previously reported non-valved device by 74% due to on-chip valve integration providing controlled and accurate assay capabilities. PMID:25310492

  9. Identification of species in tribe Brassiceae by dot-blot hybridization using species-specific ITS1 probes.

    PubMed

    Tonosaki, K; Nishio, Takeshi

    2010-10-01

    Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U's triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank. PMID:20683723

  10. Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions.

    PubMed

    Jost, J P; Saluz, H P; McEwan, I; Feavers, I M; Hughes, M; Reiber, S; Liang, H M; Vaccaro, M

    1990-01-30

    The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor. PMID:1968660