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Sample records for dna complexes probed

  1. Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes.

    PubMed

    Müller, Stefan; Neusser, Michaela; Köhler, Daniela; Cremer, Marion

    2007-01-01

    INTRODUCTIONDNA probes for fluorescence in situ hybridization (FISH) can be generated and labeled by various methods. This protocol describes the conjugation of dUTPs with haptens or fluorochromes, as well as the generation and labeling of DNA probes using those modified dUTPs. Sources of probe DNA include genomic DNA, DNA from flow-sorted chromosomes, bacterial artificial chromosomes (BACs), and cosmids. DNA amplification and labeling procedures involving degenerate oligonucleotide-primed PCR (DOP-PCR) and multiple displacement amplification (MDA) are provided. Advice is given for setting up complex probe pools, such as those containing large pools of BAC probes. Also included is a method for probe precipitation and preparation of a hybridization mix ready to be used for 3D fluorescence in situ hybridization (FISH) experiments. PMID:21357075

  2. Comparison of DNA probe and cytogenetic methods for identifying field collected Anopheles gambiae complex mosquitoes.

    PubMed

    Collins, F H; Petrarca, V; Mpofu, S; Brandling-Bennett, A D; Were, J B; Rasmussen, M O; Finnerty, V

    1988-12-01

    A recently developed DNA probe method was compared with the standard cytogenetic method for identifying the species of individual mosquitoes in the Anopheles gambiae complex. The complex consists of 6 morphologically indistinguishable sibling species that include the major African malaria vectors. Half-gravid, field collected mosquitoes were split into 2 portions: the abdomen was preserved for ovarian nurse cell cytotaxonomy and the head/thorax portion was desiccated for DNA extraction. Cytogenetic examination of the Kenya specimens showed 88 An. gambiae and 108 An. arabiensis. The Zimbabwe specimens consisted of 6 An. gambiae and 55 An. Quadriannulatus. All samples of the 3 species were polymorphic for the major chromosomal inversions previously recorded in field specimens from eastern and southern Africa, indicating that the collections reflected natural levels of intraspecific variation in the field populations sampled. Approximately 97% of the cytologically identified mosquitoes were also identified to species by the DNA probe method, and in every case the DNA probe and cytogenetic methods of species identification produced concordant results. PMID:3207175

  3. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-01

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  4. Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA**

    PubMed Central

    Baggaley, Elizabeth; Gill, Martin R; Green, Nicola H; Turton, David; Sazanovich, Igor V; Botchway, Stanley W; Smythe, Carl; Haycock, John W; Weinstein, Julia A; Thomas, Jim A

    2014-01-01

    The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging. PMID:24458590

  5. The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

    SciTech Connect

    Cooper, L.; Cooper, R.D.; Burkot, T.R. )

    1991-07-01

    Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.

  6. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  7. Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

    PubMed Central

    Bidochka, M J; Walsh, S R; Ramos, M E; Leger, R J; Silver, J C; Roberts, D W

    1995-01-01

    The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers. PMID:7574596

  8. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  9. Luminescent platinum(II) complexes with functionalized N-heterocyclic carbene or diphosphine selectively probe mismatched and abasic DNA

    PubMed Central

    Fung, Sin Ki; Zou, Taotao; Cao, Bei; Chen, Tianfeng; To, Wai-Pong; Yang, Chen; Lok, Chun-Nam; Che, Chi-Ming

    2016-01-01

    The selective targeting of mismatched DNA overexpressed in cancer cells is an appealing strategy in designing cancer diagnosis and therapy protocols. Few luminescent probes that specifically detect intracellular mismatched DNA have been reported. Here we used Pt(II) complexes with luminescence sensitive to subtle changes in the local environment and report several Pt(II) complexes that selectively bind to and identify DNA mismatches. We evaluated the complexes' DNA-binding characteristics by ultraviolet/visible absorption titration, isothermal titration calorimetry, nuclear magnetic resonance and quantum mechanics/molecular mechanics calculations. These Pt(II) complexes show up to 15-fold higher emission intensities upon binding to mismatched DNA over matched DNA and can be utilized for both detecting DNA abasic sites and identifying cancer cells and human tissue samples with different levels of mismatch repair. Our work highlights the potential of luminescent Pt(II) complexes to differentiate between normal cells and cancer cells which generally possess more aberrant DNA structures. PMID:26883164

  10. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  11. New luminescent probe based on a terbium(III) complex for studying DNA affinity of aminoalkoxy fluorenones

    NASA Astrophysics Data System (ADS)

    Yegorova, A. V.; Leonenko, I. I.; Scrypynets, Yu. V.; Antonovich, V. P.; Malzev, G. V.; Ukrainets, I. V.; Aleksandrova, D. I.

    2013-07-01

    We have studied the spectral luminescent properties of complexes of Tb(III) ions with a series of new derivatives of 2-oxo-4-hydroxyquinoline-3-carboxylic acid amides (L1-L4). We have established that DNA significantly enhances the 4f luminescence of terbium(III) in these complex compounds. We show satisfactory agreement between the logarithms of the DNA binding constants for a series of aminoalkoxy fluorenones (R1-R6), obtained using ethidium bromide and the complex Tb(III)-L1, which we propose using as a new luminescent probe for studying the affinity of drugs (prodrugs) for DNA molecules in a series of structurally similar biologically active substances.

  12. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    NASA Astrophysics Data System (ADS)

    Qin, Yangzhong; Zhong, Dongping

    2014-03-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined in nanospace but mostly highly mobile. Here, we report our studies of interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are slightly slow but similar to the surface hydration water fluctuations on picosecond time scales. Our MD simulations also show the binding interface full of water molecules and nonspecific weak interactions with protein and DNA. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.

  13. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    PubMed Central

    Qin, Yangzhong; Yang, Yi; Zhang, Luyuan; Fowler, Jason D.; Qiu, Weihong; Wang, Lijuan; Suo, Zucai; Zhong, Dongping

    2014-01-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase. PMID:24308461

  14. Application of steered molecular dynamics (SMD) to study DNA drug complexes and probing helical propensity of amino acids

    NASA Astrophysics Data System (ADS)

    Orzechowski, Marek; Cieplak, Piotr

    2005-05-01

    We present the preliminary results of two computer experiments involving the application of an external force to molecular systems. In the first experiment we simulated the process of pulling out a simple intercalator, the 9-aminoacridine molecule, from its complex with a short DNA oligonucleotide in aqueous solution. Removing a drug from the DNA is assumed to be an opposite process to the complex formation. The force and energy profiles suggest that formation of the DNA-9-aminoacridine complex is preferred when the acridine approaches the DNA from the minor groove rather than the major groove side. For a given mode of pulling the intercalation process is also shown to be nucleotide sequence dependent. In another computer experiment we performed a series of molecular dynamics simulations for stretching short, containing 15 amino acids, helical polypeptides in aqueous solution using an external force. The purpose of these simulations is to check whether this type of approach is sensitive enough to probe the sequence dependent helical propensity of short polypeptides.

  15. Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

    SciTech Connect

    Tang, K.H.; Niebuhr, M.; Aulabaugh, A.; Tsai, M.D.; /Ohio State U. /SLAC, SSRL

    2009-04-30

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.

  16. Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

    SciTech Connect

    Tang, Kuo-Hsiang; Niebuhr, Marc; Aulabaugh, Ann; Tsai, Ming-Daw

    2008-03-25

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol β-DNA complex enhances the function of Pol β.

  17. Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

    PubMed

    Golub, Eyal; Freeman, Ronit; Niazov, Angelica; Willner, Itamar

    2011-11-01

    Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition. PMID:21881641

  18. Using AFM to probe the complexation of DNA with anionic lipids mediated by Ca(2+): the role of surface pressure.

    PubMed

    Luque-Caballero, Germán; Martín-Molina, Alberto; Sánchez-Treviño, Alda Yadira; Rodríguez-Valverde, Miguel A; Cabrerizo-Vílchez, Miguel A; Maldonado-Valderrama, Julia

    2014-04-28

    Complexation of DNA with lipids is currently being developed as an alternative to classical vectors based on viruses. Most of the research to date focuses on cationic lipids owing to their spontaneous complexation with DNA. Nonetheless, recent investigations have revealed that cationic lipids induce a large number of adverse effects on DNA delivery. Precisely, the lower cytotoxicity of anionic lipids accounts for their use as a promising alternative. However, the complexation of DNA with anionic lipids (mediated by cations) is still in early stages and is not yet well understood. In order to explore the molecular mechanisms underlying the complexation of anionic lipids and DNA we proposed a combined methodology based on the surface pressure-area isotherms, Gibbs elasticity and Atomic Force Microscopy (AFM). These techniques allow elucidation of the role of the surface pressure in the complexation and visualization of the interfacial aggregates for the first time. We demonstrate that the DNA complexes with negatively charged model monolayers (DPPC/DPPS 4 : 1) only in the presence of Ca(2+), but is expelled at very high surface pressures. Also, according to the Gibbs elasticity plot, the complexation of lipids and DNA implies a whole fluidisation of the monolayer and a completely different phase transition map in the presence of DNA and Ca(2+). AFM imaging allows identification for the first time of specific morphologies associated with different packing densities. At low surface coverage, a branched net like structure is observed whereas at high surface pressure fibers formed of interfacial aggregates appear. In summary, Ca(2+) mediates the interaction between DNA and negatively charged lipids and also the conformation of the ternary system depends on the surface pressure. Such observations are important new generic features of the interaction between DNA and anionic lipids. PMID:24668321

  19. Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

    PubMed Central

    Neely, Robert K.; Daujotyte, Dalia; Grazulis, Saulius; Magennis, Steven W.; Dryden, David T. F.; Klimašauskas, Saulius; Jones, Anita C.

    2005-01-01

    DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures. PMID:16340006

  20. Metal complex interactions with DNA.

    PubMed

    Pages, Benjamin J; Ang, Dale L; Wright, Elisé P; Aldrich-Wright, Janice R

    2015-02-28

    Increasing numbers of DNA structures are being revealed using biophysical, spectroscopic and genomic methods. The diversity of transition metal complexes is also growing, as the unique contributions that transition metals bring to the overall structure of metal complexes depend on the various coordination numbers, geometries, physiologically relevant redox potentials, as well as kinetic and thermodynamic characteristics. The vast range of ligands that can be utilised must also be considered. Given this diversity, a variety of biological interactions is not unexpected. Specifically, interactions with negatively-charged DNA can arise due to covalent/coordinate or subtle non-coordinate interactions such as electrostatic attraction, groove binding and intercalation as well as combinations of all of these modes. The potential of metal complexes as therapeutic agents is but one aspect of their utility. Complexes, both new and old, are currently being utilised in conjunction with spectroscopic and biological techniques to probe the interactions of DNA and its many structural forms. Here we present a review of metal complex-DNA interactions in which several binding modes and DNA structural forms are explored. PMID:25427534

  1. Probe and method for DNA detection

    DOEpatents

    Yeh, Hsin-Chih; Werner, James Henry; Sharma, Jaswinder Kumar; Martinez, Jennifer Suzanne

    2013-07-02

    A hybridization probe containing two linear strands of DNA lights up upon hybridization to a target DNA using silver nanoclusters that have been templated onto one of the DNA strands. Hybridization induces proximity between the nanoclusters on one strand and an overhang on the other strand, which results in enhanced fluorescence emission from the nanoclusters.

  2. 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

  3. Polymer microspheres carrying fluorescent DNA probes

    NASA Astrophysics Data System (ADS)

    Chen, Xiaoyu; Dai, Zhao; Zhang, Jimei; Xu, Shichao; Wu, Chunrong; Zheng, Guo

    2010-07-01

    A polymer microspheres carried DNA probe, which was based on resonance energy transfer, was presented in this paper when CdTe quantum dots(QDs) were as energy donors, Au nanoparticles were as energy accepters and poly(4- vinylpyrindine-co-ethylene glycol dimethacrylate) microspheres were as carriers. Polymer microspheres with functional group on surfaces were prepared by distillation-precipitation polymerization when ethylene glycol dimethacrylate was as crosslinker in acetonitrile. CdTe QDs were prepared when 3-mercaptopropionic acid(MPA) was as the stabilizer in aqueous solution. Because of the hydrogen-bonding between the carboxyl groups of MPA on QDs and the pyrindine groups on the microspheres, the QDs were self-assembled onto the surfaces of microspheres. Then, the other parts of DNA probe were finished according to the classic method. The DNA detection results indicated that this novel fluorescent DNA probe system could recognize the existence of complementary target DNA or not.

  4. Development of DNA probes for Candida albicans

    SciTech Connect

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

    1988-07-01

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

  5. Fluorescent silver nanoclusters as DNA probes

    NASA Astrophysics Data System (ADS)

    Obliosca, Judy M.; Liu, Cong; Yeh, Hsin-Chih

    2013-08-01

    Fluorescent silver nanoclusters (few atoms, quantum sized) have attracted much attention as promising substitutes for conventional fluorophores. Due to their unique environmental sensitivities, new fluorescent probes have been developed based on silver nanoclusters for the sensitive and specific detection of DNA. In this review we present the recent discoveries of activatable and color-switchable properties of DNA-templated silver nanoclusters and discuss the strategies to use these new properties in DNA sensing.

  6. DNA Binding Hydroxyl Radical Probes

    PubMed Central

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2011-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA. PMID:22125376

  7. 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.

  8. SERS gene probe for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Stokes, David L.; Allain, Leonardo R.; Isola, Narayana R.; Vo-Dinh, Tuan

    2003-07-01

    We describe the development of a surface-enhanced Raman scattering gene (SERGen) probe technology for rapid screening for diseases and pathogens through DNA hybridization assays. The technology combines the use of gene probes labeled with SERS-active markers, and nanostructured metallic platforms for inducing the SERS effect. As a result, SERGen-based methods can offer the spectral selectivity and sensitivity of SERS as well as the molecular specificity of DNA sequence hybridization. Furthermore, these new probe s preclude the use of radioactive labels. As illustrated herein, SERGen probes have been used as primers in polymerase chain reaction (PCR) amplifications of specific DNA sequences, hence further boosting the sensitivity of the technology. We also describe several approaches to developing SERS-active DNA assay platforms, addressing the challenges of making the SERGen technology accessible and practical for clinical settings. The usefulness of the SERGen approach has been demonstrated in the detection of HIV, BRCA1 breast cancer, and BAX genes. There is great potential for the use of numerous SERGen probes for multiplexed detection of multiple biological targets.

  9. Probing Protein-DNA Interactions by Unzipping DNA

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2003-03-01

    Protein-DNA interactions are essential to cellular processes. In replication, transcription, recombination, DNA repair, and DNA packaging, proteins bind to DNA as activators or repressors, to recruit other proteins, or to carry out various catalytic activities. I will present Unzipping Force Analysis of Protein Association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule, we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex.

  10. Structure-based design of platinum(II) complexes as c-myc oncogene down-regulators and luminescent probes for G-quadruplex DNA.

    PubMed

    Wang, Ping; Leung, Chung-Hang; Ma, Dik-Lung; Yan, Siu-Cheong; Che, Chi-Ming

    2010-06-18

    A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G-quadruplex DNA within the c-myc gene promoter were evaluated. Complex 1, which has a flat planar 2,6-bis(benzimidazol-2-yl)pyridine (bzimpy) scaffold, was found to stabilize the c-myc G-quadruplex structure in a cell-free system. An in silico G-quadruplex DNA model has been constructed for structure-based virtual screening to develop new Pt(II)-based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit-to-lead optimization, bzimpy and related 2,6-bis(pyrazol-3-yl)pyridine (dPzPy) scaffolds containing amine side-chains emerge as the top candidates. Six of the top-scoring complexes were synthesized and their interactions with c-myc G-quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c-myc G-quadruplex. Complex 3 a ([Pt(II)L2R](+); L2=2,6-bis[1-(3-piperidinepropyl)-1H-enzo[d]imidazol-2-yl]pyridine, R=Cl) displayed the strongest inhibition in a cell-free system (IC(50)=2.2 microM) and was 3.3-fold more potent than that of 1. Complexes 3 a and 4 a ([Pt(II)L3R](+); L3=2,6-bis[1-(3-morpholinopropyl)-1H-pyrazol-3-yl]pyridine, R=Cl) were found to effectively inhibit c-myc gene expression in human hepatocarcinoma cells with IC(50) values of approximately 17 microM, whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 microM. Complexes 3 a and 4 a have a strong preference for G-quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G-quadruplex DNA with binding constants (K) of approximately 10(6)-10(7) dm(3) mol(-1), which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c-myc G-quadruplex DNA through an external end-stacking mode at

  11. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  13. Complexities of bloom dynamics in the toxic dinoflagellate Alexandrium fundyense revealed through DNA measurements by imaging flow cytometry coupled with species-specific rRNA probes

    PubMed Central

    Brosnahan, Michael L.; Farzan, Shahla; Keafer, Bruce A.; Sosik, Heidi M.; Olson, Robert J.; Anderson, Donald M.

    2013-01-01

    Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells’ nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may

  14. Complexities of bloom dynamics in the toxic dinoflagellate Alexandrium fundyense revealed through DNA measurements by imaging flow cytometry coupled with species-specific rRNA probes.

    PubMed

    Brosnahan, Michael L; Farzan, Shahla; Keafer, Bruce A; Sosik, Heidi M; Olson, Robert J; Anderson, Donald M

    2014-05-01

    Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA USA), and from a rare A. fundyense 'red tide' that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase ('2C') A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells' nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may also have

  15. Complexities of bloom dynamics in the toxic dinoflagellate Alexandrium fundyense revealed through DNA measurements by imaging flow cytometry coupled with species-specific rRNA probes

    NASA Astrophysics Data System (ADS)

    Brosnahan, Michael L.; Farzan, Shahla; Keafer, Bruce A.; Sosik, Heidi M.; Olson, Robert J.; Anderson, Donald M.

    2014-05-01

    Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA, USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells' nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore, we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may

  16. DNA/chitosan electrostatic complex.

    PubMed

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80). PMID:27050113

  17. A chiroptical photoswitchable DNA complex.

    PubMed

    Mammana, Angela; Carroll, Gregory T; Areephong, Jetsuda; Feringa, Ben L

    2011-10-13

    The interesting structural, electronic, and optical properties of DNA provide fascinating opportunities for developing nanoscale smart materials by integrating DNA with opto-electronic components. In this article we demonstrate the electrostatic binding of an amine-terminated dithienylethene (DET) molecular switch to double-stranded synthetic polynucleotides. The DET switch can undergo photochemical ring-closure and opening reactions. Circular dichroism (CD) and UV-vis spectroscopy show that both the open, 1o, and the closed, 1c, forms of the switch bind to DNA. Upon addition of DNA to a solution of 1o or 1c, the UV-vis spectrum displays a hypochromic effect, indicative of an interaction between the switch and the DNA. The chirality of the DNA double-helix is transmitted to the switching unit which displays a well-defined CD signal upon supramolecular complexation to the DNA. Additionally, the CD signal of the DNA attenuates, demonstrating that both components of the complex mutually influence each other's structure; the DNA induces chirality in the switch, and the switch modifies the structure of the DNA. Modulation of the chiroptical properties of the complex is achieved by photochemically switching the DET between its ring open and closed isomers. A pH dependence study of the binding shows that when the pH is increased the switches lose their binding ability, indicating that electrostatic interactions between protonated amines and the negatively charged phosphate backbone are the dominant driving force for binding to the DNA. A comparison of poly(deoxyguanylic-deoxycytidylic) acid [poly(dGdC)(2)] polynucleotides with poly(deoxyadenylic-deoxythymidylic) acid [poly(dAdT)(2)] shows distinct differences in the CD spectra of the complexes. PMID:21879715

  18. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  19. Monoclonals and DNA probes in diagnostic and preventative medicine

    SciTech Connect

    Gallo, R.C.; Della Povta, G.; Albertini, A.

    1987-01-01

    This book contains 24 selections. Some of the titles are: Use of DNA Probes for Prenatal and Carrier Diagnosis of Hemophilia and Fragile X Mental Retardation; The Application of DNA Probes to Diagnosis and Research of Duchenne Muscular Dystrophy: Clinical Trial, New Probes and Deletion Mapping; Molecular Genetics of the Human Collagens; Molecular Genetics of Human Steroid 21-Hydroxylase Genes; Detection of Hepatitis B Virus DNA and Hepatitis Delta Virus RNA: Implications in Diagnosis and Pathogenesis; and DNA Probes to Evaluate the Possible Association of Papovaviruses with Human Tumors.

  20. Complex DNA structures and structures of DNA complexes

    SciTech Connect

    Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

    1994-12-01

    Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

  1. Directly labeled fluorescent DNA probes for chromosome mapping

    SciTech Connect

    Marrone, B.L.; Deaven, L.L.; Chen, D.J.; Park, Min S.; MacInnes, M.A.; Salzman, G.C.; Yoshida, T.M.

    1995-12-31

    A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.

  2. Two-color spectroscopy of UV excited ssDNA complex with a single-wall nanotube (SWNT) probe: Fast nucleobase autoionization mechanism

    NASA Astrophysics Data System (ADS)

    Rotkin, Slava V.; Ignatova, Tetyana; Balaeff, Alexander; Zheng, Ming; Blades, Michael; Stoeckl, Peter

    DNA autoionization is a fundamental process wherein UV-photoexcited nucleobases dissipate energy to the environment without undergoing chemical damage. SWNT is shown to serve as a photoluminescent reporter for studying the mechanism and rates of DNA autoionization. Two-color photoluminescence (PL) spectroscopy revealed a strong SWNT PL quenching when the UV pump is resonant with the DNA absorption [Nano Research, 2015]. Semiempirical calculations of the DNA-SWNT electronic structure, combined with a Green's function theory for charge transfer, show a 20 fs autoionization rate, dominated by the hole transfer. Rate-equation analysis of the spectroscopy data confirms that the quenching rate is limited by the thermalization of the free charge carriers transferred to the nanotube reservoir. The developed approach has a great potential for monitoring DNA excitation, autoionization, and chemical damage both in vivo and in vitro. NSF ECCS-1509786 (S.V.R.,T.I.) and PHY-1359195 (P.S.), NIST and UCF facilities.

  3. Probing transient protein-mediated DNA linkages using nanoconfinement

    PubMed Central

    Roushan, Maedeh; Kaur, Parminder; Karpusenko, Alena; Countryman, Preston J.; Ortiz, Carlos P.; Fang Lim, Shuang; Wang, Hong; Riehn, Robert

    2014-01-01

    We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNA polymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches. PMID:25379073

  4. Structural Complexity of DNA Sequence

    PubMed Central

    Liou, Cheng-Yuan; Cheng, Wei-Chen; Tsai, Huai-Ying

    2013-01-01

    In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

  5. 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

  6. 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

  7. DNA probes for two Microsporidia, Nosema bombycis and Nosema costelytrae.

    PubMed

    Malone, L A; McIvor, C A

    1995-05-01

    Two DNA fragments which hybridize specifically with DNA of Nosema bombycis and Nosema costelytrae, respectively, were obtained from genomic DNA of each microsporidian species and sequenced. Neither fragment hybridized with genomic DNA from four other microsporidian isolates tested: Nosema apis, Vairimorpha sp. from cabbage white butterfly (Pieris rapae), and two isolates of Vavraia oncoperae, one from New Zealand grass grubs, Costelytrae zealandica, and another from porina caterpillars, Wiseana spp. The probe for N. bombycis did not hybridize with genomic DNA from N. costelytrae or with DNA from silkworms (Bombyx mori), the primary insect host of this microsporidium. Likewise, the probe for N. costelytrae did not hybridize with genomic DNA from N. bombycis or with DNA from grass grubs (C. zealandica). Both fragments were AT-rich (59 and 79% of total bases, respectively), had G+C/A+T ratios of 0.70 and 0.25, respectively, and represented repeated sequences dispersed throughout the genome. PMID:7745281

  8. Platinum(II) complexes as spectroscopic probes for biomolecules

    SciTech Connect

    Ratilla, E.

    1990-09-21

    The use of platinum(II) complexes as tags and probes for biomolecules is indeed advantageous for their reactivities can be selective for certain purposes through an interplay of mild reaction conditions and of the ligands bound to the platinum. The use of {sup 195}Pt NMR as a method of detecting platinum and its interactions with biomolecules was carried out with the simplest model of platinum(II) tagging to proteins. Variable-temperature {sup 195}Pt NMR spectroscopy proved useful in studying the stereodynamics of complex thioethers like methionine. The complex, Pt(trpy)Cl{sup +}, with its chromophore has a greater potential for probing proteins. It is a noninvasive and selective tag for histidine and cysteine residues on the surface of cytochrome c at pH 5. The protein derivatives obtained are separable, and the tags are easily quantitated and differentiated through the metal-to-ligand charge transfer bands which are sensitive to the environment of the tag. Increasing the pH to 7.0 led to the modification by Pt(trpy)Cl{sup +}of Arg 91 in cytochrome c. Further studies with guanidine-containing ligands as models for arginine modification by Pt(trpy)Cl{sup +} showed that guanidine can act as a terminal ligand and as a bridging ligand. Owing to the potential utility of Pt(trpy)L{sup n+} as electron dense probes of nucleic acid structure, interactions of this bis-Pt(trpy){sup 2+} complex with nucleic acids was evaluated. Indeed, the complex interacts non-covalently with nucleic acids. Its interactions with DNA are not exactly the same as those of its precedents. Most striking is its ability to form highly immobile bands of DNA upon gel electrophoresis. 232 refs.

  9. Chemical method for introducing haptens on to DNA probes

    SciTech Connect

    Keller, G.H.; Cumming, C.U.; Huang, D.P.; Manak, M.M.; Ting, R.

    1988-05-01

    The authors developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. They have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 x 10/sup -17/ mol (1 x 10/sup 6/ copies) of HBV DNA in total DNA from human serum.

  10. Commercial DNA Probes for Mycobacteria Incorrectly Identify a Number of Less Frequently Encountered Species▿ †

    PubMed Central

    Tortoli, Enrico; Pecorari, Monica; Fabio, Giuliana; Messinò, Massimino; Fabio, Anna

    2010-01-01

    Although commercially available DNA probes for identification of mycobacteria have been investigated with large numbers of strains, nothing is known about the ability of these probes to identify less frequently encountered species. We analyzed, with INNO LiPA MYCOBACTERIA (Innogenetics) and with GenoType Mycobacterium (Hein), 317 strains, belonging to 136 species, 61 of which had never been assayed before. INNO LiPA misidentified 20 taxa, the majority of which cross-reacted with the probes specific for Mycobacterium fortuitum and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. GenoType misidentified 28 taxa, most of which cross-reacted with M. intracellulare and M. fortuitum probes; furthermore, eight species were not recognized as members of the genus Mycobacterium. Among 54 strains investigated with AccuProbe (Gen-Probe), cross-reactions were detected for nine species, with the probes aiming at the M. avium complex being most involved in cross-reactions. PMID:19906898

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

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Liu, Juewen

    2014-05-01

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

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

    PubMed Central

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

    2016-01-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection–based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization. PMID:26054766

  13. Mechanisms of small molecule-DNA interactions probed by single-molecule force spectroscopy.

    PubMed

    Almaqwashi, Ali A; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C

    2016-05-19

    There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA-ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

  14. [Cu(phen)2](2+) acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor.

    PubMed

    Yang, Linlin; Li, Xiaoyu; Li, Xi; Yan, Songling; Ren, Yinna; Wang, Mengmeng; Liu, Peng; Dong, Yulin; Zhang, Chaocan

    2016-01-01

    We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)2](2+), where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)2](2+) acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)2](2+) were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10(-12) to 1 × 10(-6) M with a detection limit of 1.99 × 10(-13) M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied. PMID:26403602

  15. Detection of toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Sun, Bo; Zheng, Guo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma gondii is a microscopic parasite that may infect humans, so there is an increasing concern on the early detection of latent Toxoplasma gondii infection in recent years. We currently report a rapid and sensitive method for Toxoplasma gondii based on molecular beacon (MB) probe. The probe based on fluorescence resonance energy transfer (FRET) with a stem-loop DNA oligonucleotide was labeled with CdTe/ZnS quantum dots (energy donor) at 5' end and BHQ-2 (energy acceptor) at 3' end, respectively. The probe was synthesized in PBS buffer at pH 8.2, room temperature for 24 h. Then target DNA was injected under the condition of 37°C, hybridization for 2 h, in Tris-HCl buffer. The data from fluorescence spectrum (FS) showed that ca 65% of emitted fluorescence was quenched, and about 50% recovery of fluorescence intensity was observed after adding target DNA, which indicated that the target DNA was successfully detected by MB probe. The detecting limitation was determined as ca 5 nM. Moreover, specificity of the probe was investigated by adding target DNA with one-base-pair mismatch, the low fluorescence recovery indicated the high specificity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  16. Probing the Mechanical Unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Voulgarakis, N. K.; Redondo, A.; Bishop, A. R.; Rasmussen, K. Ø.

    2006-06-01

    A study of the micromechanical unzipping of DNA in the framework of the Peyrard-Bishop-Dauxois model is presented. We introduce a Monte Carlo technique that allows accurate determination of the dependence of the unzipping forces on unzipping speed and temperature. Our findings agree quantitatively with experimental results for homogeneous DNA, and for λ-phage DNA we reproduce the recently obtained experimental force-temperature phase diagram. Finally, we argue that there may be fundamental differences between in vivo and in vitro DNA unzipping.

  17. APPLICATIONS FOR DNA PROBES IN BIODEGRADATION RESEARCH

    EPA Science Inventory

    The use of DNA:DNA hybridization technology in biodegradation studies is investigated. The rate constants for sediments exposed to synthetic oils could be calculated from the NAH(1+) genotypes and this approach would be useful in predicting the kinetics of aromatic hydrocarbon de...

  18. Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

    PubMed Central

    Almaqwashi, Ali A.; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C.

    2016-01-01

    There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

  19. Probing the elastic limit of DNA bending

    PubMed Central

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Sharp bending of double-stranded DNA (dsDNA) plays an essential role in genome structure and function. However, the elastic limit of dsDNA bending remains controversial. Here, we measured the opening rates of small dsDNA loops with contour lengths ranging between 40 and 200 bp using single-molecule Fluorescence Resonance Energy Transfer. The relationship of loop lifetime to loop size revealed a critical transition in bending stress. Above the critical loop size, the loop lifetime changed with loop size in a manner consistent with elastic bending stress, but below it, became less sensitive to loop size, indicative of softened dsDNA. The critical loop size increased from ∼60 bp to ∼100 bp with the addition of 5 mM magnesium. We show that our result is in quantitative agreement with the kinkable worm-like chain model, and furthermore, can reproduce previously reported looping probabilities of dsDNA over the range between 50 and 200 bp. Our findings shed new light on the energetics of sharply bent dsDNA. PMID:25122748

  20. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    NASA Astrophysics Data System (ADS)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-06-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  1. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation.

    PubMed

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-12-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met-p16). The probe, paired with Met-p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases. PMID:27325520

  2. Probing the mechanical unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Voulgarakis, Nikos K.; Bishop, Alan R.; Rasmussen, Kim O.

    2006-03-01

    Recent advances in single-molecule force spectroscopy have made a systematic study of local melting in DNA possible. This provide new insight into important biological processes as replication and transcription. In this work, we present an extensive study of the micromechanical unzipping of DNA in the framework of the Peyrard-Bishop-Dauxois (PBD) model. The force required to separate the doubled strand is derived through analysis of the force-extension curve, while an estimation of the nucleation bubble size of the unzipping process is obtained by the distribution of the rapture force. Our findings are in very good agreement with existing experimental results; for example the force-temperature phase diagram obtained by the PBD model agrees excellently with recent constant-force experimental measurements of the lambda-phage DNA. Fundamental differences between the in vivo and vitro DNA unzipping, as predicted by the PBD model, are also discussed.

  3. Direct electronic probing of biological complexes formation

    NASA Astrophysics Data System (ADS)

    Macchia, Eleonora; Magliulo, Maria; Manoli, Kyriaki; Giordano, Francesco; Palazzo, Gerardo; Torsi, Luisa

    2014-10-01

    Functional bio-interlayer organic field - effect transistors (FBI-OFET), embedding streptavidin, avidin and neutravidin as bio-recognition element, have been studied to probe the electronic properties of protein complexes. The threshold voltage control has been achieved modifying the SiO2 gate diaelectric surface by means of the deposition of an interlayer of bio-recognition elements. A threshold voltage shift with respect to the unmodified dielectric surface toward more negative potential values has been found for the three different proteins, in agreement with their isoelectric points. The relative responses in terms of source - drain current, mobility and threshold voltage upon exposure to biotin of the FBI-OFET devices have been compared for the three bio-recognition elements.

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

    NASA Astrophysics Data System (ADS)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

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

  5. Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end.

    PubMed

    Yoo, S; Kimzey, A; Dynan, W S

    1999-07-01

    Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively. PMID:10391954

  6. 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

  7. Probing the microscopic flexibility of DNA from melting temperatures

    NASA Astrophysics Data System (ADS)

    Weber, Gerald; Essex, Jonathan W.; Neylon, Cameron

    2009-10-01

    The microscopic flexibility of DNA is a key ingredient for understanding its interaction with proteins and drugs but is still poorly understood and technically challenging to measure. Several experimental methods probe very long DNA samples, but these miss local flexibility details. Others mechanically disturb or modify short molecules and therefore do not obtain flexibility properties of unperturbed and pristine DNA. Here, we show that it is possible to extract very detailed flexibility information about unmodified DNA from melting temperatures with statistical physics models. We were able to retrieve, from published melting temperatures, several established flexibility properties such as the presence of highly flexible TATA regions of genomic DNA and support recent findings that DNA is very flexible at short length scales. New information about the nanoscale Na+ concentration dependence of DNA flexibility was determined and we show the key role of ApT and TpA steps when it comes to ion-dependent flexibility and melting temperatures.

  8. Robust 3D DNA FISH using directly labeled probes.

    PubMed

    Bolland, Daniel J; King, Michelle R; Reik, Wolf; Corcoran, Anne E; Krueger, Christel

    2013-01-01

    3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions. PMID:23978815

  9. Robust 3D DNA FISH Using Directly Labeled Probes

    PubMed Central

    Bolland, Daniel J.; King, Michelle R.; Reik, Wolf; Corcoran, Anne E.; Krueger, Christel

    2013-01-01

    3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions. PMID:23978815

  10. Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

    SciTech Connect

    Xia, Shuangluo; Christian, Thomas D.; Wang, Jimin; Konigsberg, William H.

    2012-09-17

    Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10{sup 2}-10{sup 3}-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10{sup 2}-10{sup 3}-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n-2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.

  11. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  12. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  13. Diamondoids as DNA methylation and mutation probes

    NASA Astrophysics Data System (ADS)

    Sivaraman, Ganesh; Fyta, Maria

    2014-10-01

    In a recent study we proposed derivatives of lower diamondoids as novel biosensors, as well as possible functionalisation candidates of solid-state nanopores for DNA sequencing. A qualitative analysis has shown the abilities of these molecules to distinguish among different DNA nucleobases. In this letter, we extend the analysis and consider also methylated and mutated nucleobases, often being an indication of genetic diseases. Based on the bonding characteristics of these modified nucleobases to a diamondoid derivative, as well as their electronic properties we could reveal the ability of the diamondoid to clearly distinguish the regular from the modified nucleobases. The results show a clear indication that transport properties along these molecules would give distinct current signals.

  14. 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

  15. Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

    SciTech Connect

    Arfin, Najmul; Yadav, Avinash Chand; Bohidar, H. B.

    2013-11-15

    The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement 〈R{sup 2}〉 exhibits a scaling with time as 〈R{sup 2}〉 ∼ t{sup α}, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ∼ t{sup β}/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

  16. Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

    NASA Astrophysics Data System (ADS)

    Arfin, Najmul; Yadav, Avinash Chand; Bohidar, H. B.

    2013-11-01

    The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement ⟨R2⟩ exhibits a scaling with time as ⟨R2⟩ ˜ tα, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ˜ tβ/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

  17. 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.

  18. Probing DNA-lipid membrane interactions with a lipopeptide nanopore.

    PubMed

    Bessonov, Andrey; Takemoto, Jon Y; Simmel, Friedrich C

    2012-04-24

    Association of DNA molecules with lipid bilayer membranes is of considerable interest for a large variety of applications in biotechnology. Here we introduce syringomycin E (SRE), a small pore-forming lipopeptide produced by the bacterium Pseudomonas syringae, as a facile sensor for the detection of DNA interactions with lipid membranes. SRE forms highly reproducible pores in cellular and artificial membranes. The pore structure involves bilayer lipids, which have a pronounced influence on open channel conductance and gating. SRE channels act as ionic diodes that serve as current rectifiers sensitive to the charge of the bilayer. We employ this intrinsic property to electronically monitor the association of DNA molecules with the membrane in a variety of different settings. We show that SRE can be used for quantitatively probing electrostatic interactions of DNA and DNA-cholesterol conjugates with a lipid membrane. Furthermore, we demonstrate that SRE channels allow monitoring of hybridization reactions between lipid-anchored probe strands and complementary strands in solution. In the presence of double-stranded DNA, SRE channels display a particularly high degree of rectification. Finally, the formation of multilayered structures assembled from poly-(L)-lysine and DNA oligonucleotides on the membrane was precisely monitored with SRE. PMID:22424398

  19. DNA probes for the identification of Haemophilus ducreyi.

    PubMed

    Parsons, L M; Shayegani, M; Waring, A L; Bopp, L H

    1989-07-01

    Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32P, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 10(7) to 6.0 x 10(7) CFU was tested. When 10(5) to 10(6) CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 10(4) CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 10(3) CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen. PMID:2788660

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

    SciTech Connect

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

    2008-12-16

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

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

    SciTech Connect

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

    2008-12-04

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

  2. Photonic Crystal Biosensor with In-Situ Synthesized DNA Probes for Enhanced Sensitivity

    SciTech Connect

    Hu, Shuren; Zhao, Y.; Retterer, Scott T; Kravchenko, Ivan I; Weiss, Sharon

    2013-01-01

    We report on a nearly 8-fold increase in multi-hole defect photonic crystal biosensor response by incorporating in-situ synthesis of DNA probes, as compared to the conventional functionalization method employing pre-synthesized DNA probe immobilization.

  3. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    SciTech Connect

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

    2009-01-30

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

  4. Luminescent Iridium(III) Complex Labeled DNA for Graphene Oxide-Based Biosensors.

    PubMed

    Zhao, Qingcheng; Zhou, Yuyang; Li, Yingying; Gu, Wei; Zhang, Qi; Liu, Jian

    2016-02-01

    There has been growing interest in utilizing highly photostable iridium(III) complexes as new luminescent probes for biotechnology and life science. Herein, iridium(III) complex with carboxyl group was synthesized and activated with N-hydroxysuccinimide, followed by tagging to the amino terminate of single-stranded DNA (ssDNA). The Ir-ssDNA probe was further combined with graphene oxide (GO) nanosheets to develop a GO-based biosensor for target ssDNA detection. The quenching efficiency of GO, and the photostability of iridium(III) complex and GO-Ir-ssDNA biosensor, were also investigated. On the basis of the high luminescence quenching efficiency of GO toward iridium(III) complex, the GO-Ir-ssDNA biosensor exhibited minimal background signals, while strong emission was observed when Ir-ssDNA desorbed from GO nanosheets and formed a double helix with the specific target, leading to a high signal-to-background ratio. Moreover, it was found that luminescent intensities of iridium(III) complex and GO-Ir-ssDNA biosensor were around 15 and 3 times higher than those of the traditional carboxyl fluorescein (FAM) dye and the GO-FAM-ssDNA biosensor after UV irradiation, respectively. Our study suggested the sensitive and selective Ir-ssDNA probe was suitable for the development of highly photostable GO-based detection platforms, showing promise for application beyond the OLED (organic light emitting diode) area. PMID:26753824

  5. Melanesian mtDNA Complexity

    PubMed Central

    Friedlaender, Jonathan S.; Friedlaender, Françoise R.; Hodgson, Jason A.; Stoltz, Matthew; Koki, George; Horvat, Gisele; Zhadanov, Sergey; Schurr, Theodore G.; Merriwether, D. Andrew

    2007-01-01

    Melanesian populations are known for their diversity, but it has been hard to grasp the pattern of the variation or its underlying dynamic. Using 1,223 mitochondrial DNA (mtDNA) sequences from hypervariable regions 1 and 2 (HVR1 and HVR2) from 32 populations, we found the among-group variation is structured by island, island size, and also by language affiliation. The more isolated inland Papuan-speaking groups on the largest islands have the greatest distinctions, while shore dwelling populations are considerably less diverse (at the same time, within-group haplotype diversity is less in the most isolated groups). Persistent differences between shore and inland groups in effective population sizes and marital migration rates probably cause these differences. We also add 16 whole sequences to the Melanesian mtDNA phylogenies. We identify the likely origins of a number of the haplogroups and ancient branches in specific islands, point to some ancient mtDNA connections between Near Oceania and Australia, and show additional Holocene connections between Island Southeast Asia/Taiwan and Island Melanesia with branches of haplogroup E. Coalescence estimates based on synonymous transitions in the coding region suggest an initial settlement and expansion in the region at ∼30–50,000 years before present (YBP), and a second important expansion from Island Southeast Asia/Taiwan during the interval ∼3,500–8,000 YBP. However, there are some important variance components in molecular dating that have been overlooked, and the specific nature of ancestral (maternal) Austronesian influence in this region remains unresolved. PMID:17327912

  6. Chemical Biology Probes from Advanced DNA-encoded Libraries.

    PubMed

    Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

    2016-02-19

    The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs. PMID:26820267

  7. Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

    PubMed

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W; Palleschi, Giuseppe; Ricci, Francesco

    2010-11-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  8. Structure of DNA-liposome complexes

    SciTech Connect

    Lasic, D.D.; Strey, H.; Podgornik, R.; Stuart, M.C.A.; Frederik, P.M.

    1997-01-29

    Despite numerous studies and commericially available liposome kits, however, the structure of DNA-cationic liposome complexes is still not yet well understood. We have investigated the structure of these complexes using high-resolution cryo electron microscopy (EM) and small angle X-ray scattering (SAXS). 14 refs., 3 figs.

  9. The Structure of DNA within Cationic Lipid/DNA Complexes

    PubMed Central

    Braun, Chad S.; Jas, Gouri S.; Choosakoonkriang, Sirirat; Koe, Gary S.; Smith, Janet G.; Middaugh, C. Russell

    2003-01-01

    The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed. PMID:12547792

  10. Toxoplasma gondii DNA detection with a magnetic molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma Gondii infection is widespread in humans worldwide and reported infection rates range from 3%-70%, depending on the populations or geographic areas, and it has been recognized as a potential food safety hazard in our daily life. A magnetic molecular beacon probe (mMBP), based on theory of fluorescence resonance energy transfer (FRET), was currently reported to detect Toxoplasma Gondii DNA. Nano-sized Fe3O4 were primarily prepared by coprecipitation method in aqueous phase with NaOH as precipitator, and was used as magnetic core. The qualified coreshell magnetic quantum dots (mQDs), i.e. CdTe(symbol)Fe3O4, were then achieved by layer-by-layer method when mol ratio of Fe3O4/CdTe is 1/3, pH at 6.0, 30 °C, and reactant solution was refluxed for 30 min, the size of mQDs were determined to be 12-15 nm via transmission electron microscopy (TEM). Over 70% overlap between emission spectrum of mQDs and absorbance spectrum of BHQ-2 was observed, this result suggests the synthesized mQDs and BHQ-2 can be utilized as energy donor and energy acceptor, respectively. The sensing probe was fabricated and a stem-loop Toxoplasma Gondii DNA oligonucleotide was labeled with mQDs at the 5' end and BHQ-2 at 3' end, respectively. Target Toxoplasma gondii DNA was detected under conditions of 37 °C, hybridization for 2h, at pH8.0 in Tris-HCl buffer. About 30% recovery of fluorescence intensity was observed via fluorescence spectrum (FS) after the Toxoplasma gondii DNA was added, which suggested that the Toxoplasma Gondii DNA was successfully detected. Specificity investigation of the mMBP indicated that relative low recovery of fluorescence intensity was obtained when the target DNA with one-base pair mismatch was added, this result indicated the high specificity of the sensing probe. Our research simultaneously indicated that mMBP can be conveniently separated from the unhybridized stem-loop DNA and target DNA, which will be meaningful in DNA sensing and purification process.

  11. Sequence of a DNA probe specific for Anopheles quadrimaculatus species A (Diptera: Culicidae).

    PubMed

    Johnson, D W; Cockburn, A F; Seawright, J A

    1993-09-01

    The nucleotide sequence was determined for a portion of a 12-kb genomic DNA clone specific for Anopheles quadrimaculatus species A. Four short, internally repeated sequences were identified. Synthetic oligonucleotide probes were prepared based on these four repeats. The oligonucleotides are highly specific and can be reliably used to separate individuals of An. quadrimaculatus species A from members of other species of the complex. PMID:8254645

  12. SMC complexes: from DNA to chromosomes.

    PubMed

    Uhlmann, Frank

    2016-07-01

    SMC (structural maintenance of chromosomes) complexes - which include condensin, cohesin and the SMC5-SMC6 complex - are major components of chromosomes in all living organisms, from bacteria to humans. These ring-shaped protein machines, which are powered by ATP hydrolysis, topologically encircle DNA. With their ability to hold more than one strand of DNA together, SMC complexes control a plethora of chromosomal activities. Notable among these are chromosome condensation and sister chromatid cohesion. Moreover, SMC complexes have an important role in DNA repair. Recent mechanistic insight into the function and regulation of these universal chromosomal machines enables us to propose molecular models of chromosome structure, dynamics and function, illuminating one of the fundamental entities in biology. PMID:27075410

  13. Detection of Toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Zhou, Cun; Xu, Shichao; Yang, Juan; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Sun, Shuqing; Feng, Teilin; Zi, Yan; Liang, Chu; Luo, Hao

    2009-07-01

    Toxoplasma gondii is a kind of microscopic parasite that may infect humans, and there are increasing concerns on the early detection of latent Toxoplasma gondii infection in recent years. This research highlights a new type of molecular beacon (MB) fluorescent probe for Toxoplasma DNA testing. We combined high-efficiency fluorescent inorganic core-shell quantum dots-CdTe/ZnS (as fluorescent energy donor) and BHQ-2 (energy acceptor) to the single-strand DNA of Toxoplasma gondii, and a molecular beacon sensing system based on fluorescence resonance energy transfer (FRET) was achieved. Core-shell quantum dots CdTe/ZnS was firstly prepared in aqueous solution, and the influencing factor of its fluorescent properties, including CdTe/Na2S/Zn(CH3COO)2 (v/v), dependence of reaction time, temperature, and pH, is investigated systematically. The synthesized quantum dots and molecular beacon were characterized by transmission electron microscopy (TEM), ultraviolet-visible spectrophotometer (UV-vis), fluorescent spectrophotometer (FS), respectively. The TEM results showed that CdTe/ZnS core-shell quantum dots is ~11nm in size, and the quantum dots is water-soluble well. The sensing ability of target DNA of assembled MB was investigated, and results showed that the target Toxoplasma gonddi DNA can be successfully detected by measuring the change of fluorescence intensity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  14. 6MAP, a fluorescent adenine analogue, is a probe of base flipping by DNA photolyase.

    PubMed

    Yang, Kongsheng; Matsika, Spiridoula; Stanley, Robert J

    2007-09-01

    Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the Förster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra. PMID:17696385

  15. Epidemiological typing of Moraxella catarrhalis by using DNA probes.

    PubMed Central

    Beaulieu, D; Scriver, S; Bergeron, M G; Low, D E; Parr, T R; Patterson, J E; Matlow, A; Roy, P H

    1993-01-01

    Small-fragment restriction enzyme analysis and DNA-DNA hybridization were used to compare 60 strains of Moraxella catarrhalis isolated from various geographic locations. Restriction enzyme analysis with HaeIII resulted in 46 different patterns, 7 of which were shared by more than one isolate. Hybridizations with two DNA probes resulted in 18 different patterns, 11 of which were shared by more than one isolate. Strains with the same restriction enzyme pattern always had the same hybridization pattern. However, of the 50 strains that shared the 11 hybridization patterns, 39 could be further differentiated by restriction enzyme analysis. We found that hybridization is a method that is specific for the epidemiological typing of M. catarrhalis, but because of limited sensitivity, combination with small-fragment restriction enzyme analysis may be necessary to better determine the relatedness of strains. Images PMID:8096219

  16. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  17. Complex quantum networks as structured environments: engineering and probing

    NASA Astrophysics Data System (ADS)

    Nokkala, Johannes; Galve, Fernando; Zambrini, Roberta; Maniscalco, Sabrina; Piilo, Jyrki

    2016-05-01

    We consider structured environments modeled by bosonic quantum networks and investigate the probing of their spectral density, structure, and topology. We demonstrate how to engineer a desired spectral density by changing the network structure. Our results show that the spectral density can be very accurately detected via a locally immersed quantum probe for virtually any network configuration. Moreover, we show how the entire network structure can be reconstructed by using a single quantum probe. We illustrate our findings presenting examples of spectral densities and topology probing for networks of genuine complexity.

  18. Complex quantum networks as structured environments: engineering and probing.

    PubMed

    Nokkala, Johannes; Galve, Fernando; Zambrini, Roberta; Maniscalco, Sabrina; Piilo, Jyrki

    2016-01-01

    We consider structured environments modeled by bosonic quantum networks and investigate the probing of their spectral density, structure, and topology. We demonstrate how to engineer a desired spectral density by changing the network structure. Our results show that the spectral density can be very accurately detected via a locally immersed quantum probe for virtually any network configuration. Moreover, we show how the entire network structure can be reconstructed by using a single quantum probe. We illustrate our findings presenting examples of spectral densities and topology probing for networks of genuine complexity. PMID:27230125

  19. Complex quantum networks as structured environments: engineering and probing

    PubMed Central

    Nokkala, Johannes; Galve, Fernando; Zambrini, Roberta; Maniscalco, Sabrina; Piilo, Jyrki

    2016-01-01

    We consider structured environments modeled by bosonic quantum networks and investigate the probing of their spectral density, structure, and topology. We demonstrate how to engineer a desired spectral density by changing the network structure. Our results show that the spectral density can be very accurately detected via a locally immersed quantum probe for virtually any network configuration. Moreover, we show how the entire network structure can be reconstructed by using a single quantum probe. We illustrate our findings presenting examples of spectral densities and topology probing for networks of genuine complexity. PMID:27230125

  20. Homo- and Heterobimetallic Ruthenium(II) and Osmium(II) Complexes Based on a Pyrene-Biimidazolate Spacer as Efficient DNA-Binding Probes in the Near-Infrared Domain.

    PubMed

    Mardanya, Sourav; Karmakar, Srikanta; Mondal, Debiprasad; Baitalik, Sujoy

    2016-04-01

    We report in this work a new family of homo- and heterobimetallic complexes of the type [(bpy)2M(Py-Biimz)M'(II)(bpy)2](2+) (M = M' = Ru(II) or Os(II); M = Ru(II) and M' = Os(II)) derived from a pyrenyl-biimidazole-based bridge, 2-imidazolylpyreno[4,5-d]imidazole (Py-BiimzH2). The homobimetallic Ru(II) and Os(II) complexes were found to crystallize in monoclinic form with space group P21/n. All the complexes exhibit strong absorptions throughout the entire UV-vis region and also exhibit luminescence at room temperature. For osmium-containing complexes (2 and 3) both the absorption and emission band stretched up to the NIR region and thus afford more biofriendly conditions for probable applications in infrared imaging and phototherapeutic studies. Detailed luminescence studies indicate that the emission originates from the respective (3)MLCT excited state mainly centered in the [M(bpy)2](2+) moiety of the complexes and is only slightly affected by the pyrene moiety. The bimetallic complexes show two successive one-electron reversible metal-centered oxidations in the positive potential window and several reduction processes in the negative potential window. An efficient intramolecular electronic energy transfer is found to occur from the Ru center to the Os-based component in the heterometallic dyad. The binding studies of the complexes with DNA were thoroughly studied through different spectroscopic techniques such as UV-vis absorption, steady-state and time-resolved emission, circular dichroism, and relative DNA binding study using ethidium bromide. The intercalative mode of binding was suggested to be operative in all cases. Finally, computational studies employing DFT and TD-DFT were also carried out to interpret the experimentally observed absorption and emission bands of the complexes. PMID:27011117

  1. Transcription initiation complex structures elucidate DNA opening.

    PubMed

    Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

    2016-05-19

    Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts. PMID:27193681

  2. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  3. Intercalation processes of copper complexes in DNA

    PubMed Central

    Galindo-Murillo, Rodrigo; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Cheatham, Thomas E.; Cortés-Guzmán, Fernando

    2015-01-01

    The family of anticancer complexes that include the transition metal copper known as Casiopeínas® shows promising results. Two of these complexes are currently in clinical trials. The interaction of these compounds with DNA has been observed experimentally and several hypotheses regarding the mechanism of action have been developed, and these include the generation of reactive oxygen species, phosphate hydrolysis and/or base-pair intercalation. To advance in the understanding on how these ligands interact with DNA, we present a molecular dynamics study of 21 Casiopeínas with a DNA dodecamer using 10 μs of simulation time for each compound. All the complexes were manually inserted into the minor groove as the starting point of the simulations. The binding energy of each complex and the observed representative type of interaction between the ligand and the DNA is reported. With this extended sampling time, we found that four of the compounds spontaneously flipped open a base pair and moved inside the resulting cavity and four compounds formed stacking interactions with the terminal base pairs. The complexes that formed the intercalation pocket led to more stable interactions. PMID:25958394

  4. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    PubMed Central

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2011-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe’s redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  5. 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

  6. Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes.

    PubMed Central

    Ni, H.; Knight, A. I.; Cartwright, K. A.; McFadden, J. J.

    1992-01-01

    The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence IS1106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15:P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion, were found to be derived from two distinct lineages within the B15:P1.16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningococcus do not necessarily reflect true genetic relationships. Images Fig. 1 PMID:1356820

  7. 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.

  8. 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

  9. Geant4-DNA simulations using complex DNA geometries generated by the DnaFabric tool

    NASA Astrophysics Data System (ADS)

    Meylan, S.; Vimont, U.; Incerti, S.; Clairand, I.; Villagrasa, C.

    2016-07-01

    Several DNA representations are used to study radio-induced complex DNA damages depending on the approach and the required level of granularity. Among all approaches, the mechanistic one requires the most resolved DNA models that can go down to atomistic DNA descriptions. The complexity of such DNA models make them hard to modify and adapt in order to take into account different biological conditions. The DnaFabric project was started to provide a tool to generate, visualise and modify such complex DNA models. In the current version of DnaFabric, the models can be exported to the Geant4 code to be used as targets in the Monte Carlo simulation. In this work, the project was used to generate two DNA fibre models corresponding to two DNA compaction levels representing the hetero and the euchromatin. The fibres were imported in a Geant4 application where computations were performed to estimate the influence of the DNA compaction on the amount of calculated DNA damage. The relative difference of the DNA damage computed in the two fibres for the same number of projectiles was found to be constant and equal to 1.3 for the considered primary particles (protons from 300 keV to 50 MeV). However, if only the tracks hitting the DNA target are taken into account, then the relative difference is more important for low energies and decreases to reach zero around 10 MeV. The computations were performed with models that contain up to 18,000 DNA nucleotide pairs. Nevertheless, DnaFabric will be extended to manipulate multi-scale models that go from the molecular to the cellular levels.

  10. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  11. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  12. [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

  13. Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein.

    PubMed

    Yoo, S; Dynan, W S

    1999-12-15

    Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested. PMID:10572166

  14. Potentiostatic deposition of DNA for scanning probe microscopy.

    PubMed Central

    Lindsay, S M; Tao, N J; DeRose, J A; Oden, P I; Lyubchenko YuL; Harrington, R E; Shlyakhtenko, L

    1992-01-01

    We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially. Images FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:1617139

  15. Strain differentiation of Onchocerca volvulus from Uganda using DNA probes.

    PubMed

    Fischer, P; Bamuhiiga, J; Kilian, A H; Büttner, D W

    1996-04-01

    Polymerase chain reaction (PCR) combined with non-radioactive DNA hybridization was applied for the detection and characterization of a 150 bp tandem repeat of Onchocerca volvulus. DNA of worms from western Uganda was amplified and then probed with a digoxygenin-labelled oligonucleotide, specific for the forest form of O. volvulus and compared to samples from various African countries. Hybridization was only observed with PCR products from the forest in Liberia, south-eastern Ghana, Benin and southern Cameroon, but not with worms from Uganda or the savannah in Burkina Faso and northern Ghana. A nested PCR using primers derived form the forest form-specific DNA sequence confirmed these results. Morphometric studies revealed length differences between the microfilariae of Ugandan O. volvulus to those of West Africa, especially to those of the savannah in Burkina Faso. It is concluded that the forest/savannah classification of O. volvulus from West Africa is not suitable for Simulium neavei-transmitted O. volvulus from Uganda. PMID:8935951

  16. DNA based computing for understanding complex shapes.

    PubMed

    Ullah, A M M Sharif; D'Addona, Doriana; Arai, Nobuyuki

    2014-03-01

    This study deals with a computing method called DNA based computing (DBC) that takes inspiration from the Central Dogma of Molecular Biology. The proposed DBC uses a set of user-defined rules to create a DNA-like sequence from a given piece of problem-relevant information (e.g., image data) in a dry-media (i.e., in an ordinary computer). It then uses another set of user-defined rules to create an mRNA-like sequence from the DNA. Finally, it uses the genetic code to translate the mRNA (or directly the DNA) to a protein-like sequence (a sequence of amino acids). The informational characteristics of the protein (entropy, absence, presence, abundance of some selected amino acids, and relationships among their likelihoods) can be used to solve problems (e.g., to understand complex shapes from their image data). Two case studies ((1) fractal geometry generated shape of a fern-leaf and (2) machining experiment generated shape of the worn-zones of a cutting tool) are presented elucidating the shape understanding ability of the proposed DBC in the presence of a great deal of variability in the image data of the respective shapes. The implication of the proposed DBC from the context of Internet-aided manufacturing system is also described. Further study can be carried out in solving other complex computational problems by using the proposed DBC and its derivatives. PMID:24447435

  17. Scrutinizing the DNA damaging and antimicrobial abilities of triazole appended metal complexes.

    PubMed

    Utthra, Ponnukalai Ponya; Pravin, Narayanaperumal; Raman, Natarajan

    2016-05-01

    New mononuclear transition metal complexes 1-12 bearing the bioactive triazole analogues were synthesized and characterized by elemental analysis and spectroscopic techniques. The interaction of calf thymus DNA (CT-DNA) with the synthesized compounds was studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques. The entire DNA binding results suggested the intercalative mode of binding for the synthesized compounds. Interestingly, the binding strength of the complexes is found to be greater than that of the free ligands. Among the complexes explored, complex 5 reveals strong hypochromism and a slight red shift as compared to the other complexes highlighting its higher DNA binding propensity. The intrinsic binding constant values of the complexes compared to cisplatin reveal that all the complexes are greater in magnitude than that of cisplatin. Fluorescence titrations show that the Cu(II) complexes have the ability to displace DNA-bound ethidium bromide. Also, these compounds induce cleavage in pBR322 plasmid DNA as indicated in gel electrophoresis and exhibit excellent nuclease activity in the presence of H2O2. Moreover, the complexes were screened for in vitro antimicrobial activity along with free ligands and solvent control. The outcome is that the complexes possess good activity than the free ligands. These complexes may have further scope in developing them into antimicrobial drugs and DNA probes. PMID:26971279

  18. DNA Photonics — Probing Light-Induced Dynamics in DNA on the Femtosecond Timescale

    NASA Astrophysics Data System (ADS)

    Wang, Qiang; Fiebig, Torsten

    In Chap. 10, Wang and Fiebig discuss about a new field, DNA photonics that is important to understand the role of DNA as a functional building block in molecular nanoscale devices, and is also expected to shed light on the complex interactions between structural and electronic properties of DNA. The latter is important for biomedical applications such as DNA-targeted drug design. In this chapter, the authors present experimental data from several different classes of functionalized DNA systems and illustrate the relationship between the structural dynamics and charge injection/migration using state-of-the art femtosecond broadband spectroscopy. They also highlight the importance of the initial electronic excitation for modelling electron transfer rates and point out that ultrafast electronic energy migration, dissipation, and (de)localization must be included into the theoretical description of light-induced dynamics in DNA.

  19. Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

    SciTech Connect

    Hastings, Adam F.; Otting, Gottfried; Folmer, Rutger H.A.; Duggin, Iain G.; Wake, R. Gerry; Wilce, Matthew C.J.; Wilce, Jacqueline A. . E-mail: Jackie.Wilce@med.monash.edu.au

    2005-09-23

    Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in {sup 1}H-{sup 15}N correlation spectra of a {sup 15}N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the {sup 1}H-{sup 15}N correlations was achieved using a suite of triple resonance NMR experiments with {sup 15}N,{sup 13}C,70% {sup 2}H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to {sup 1}H-{sup 15}N spectra revealed that the N-termini, {alpha}3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

  20. Thermally forced transitions of DNA-CTMA complex microstructure

    NASA Astrophysics Data System (ADS)

    Nizioł, Jacek; Ekiert, Robert; Śniechowski, Maciej; Słomiany, Magdalena; Marzec, Mateusz M.

    2016-06-01

    DNA complexed with amphiphilic cationic surfactants is a new class of optical material. In this work DNA and its complex with cetyltrimetyl ammonium chloride were thermally annealed. X-ray diffractometry revealed irreversible changes of DNA-CTMA microstructure. The new microstucture that appeared in result of the first heating course was stable, despite the further thermal annealing. Agarose gel electrophoresis indicated fundamental differences between thermally treated native DNA and DNA-CTMA complex.

  1. Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction.

    PubMed Central

    Beggs, M L; Cave, M D; Marlowe, C; Cloney, L; Duck, P; Eisenach, K D

    1996-01-01

    Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110. PMID:8940435

  2. DNA probes for papillomavirus strains readied for cervical cancer screening

    SciTech Connect

    Merz, B.

    1988-11-18

    New Papillomavirus tests are ready to come to the aid of the standard Papanicolauo test in screening for cervical cancer. The new tests, which detect the strains of human papillomavirus (HPV) most commonly associated with human cervical cancer, are designed to be used as an adjunct to rather than as a replacement for the Papanicolaou smears. Their developers say that they can be used to indicated a risk of developing cancer in women whose Papanicolaou smears indicate mild cervical dysplasia, and, eventually, to detect papillomavirus infection in normal Papanicolaou smears. The rationale for HPV testing is derived from a growing body of evidence that HPV is a major factor in the etiology of cervical cancer. Three HPV tests were described recently in Chicago at the Third International Conference on Human Papillomavirus and Squamous Cervical Cancer. Each relies on DNA probes to detect the presence of papillomavirus in cervical cells and/or to distinguish the strain of papillomavirus present.

  3. Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.

    PubMed

    Anderson, Brooke A; Karmakar, Saswata; Hrdlicka, Patrick J

    2015-01-01

    Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine and 2'-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms. PMID:26230684

  4. Detection and classification of Trypanosoma cruzi by DNA hybridization with nonradioactive probes.

    PubMed

    Solari, A; Venegas, J; Gonzalez, E; Vasquez, C

    1991-01-01

    Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes. PMID:1667933

  5. Method and apparatus for synthesis of arrays of DNA probes

    DOEpatents

    Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.; Singh-Gasson, Sangeet; Green, Roland

    2002-04-23

    The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, which may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.

  6. Nanoscale structure of protamine/DNA complexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Motta, Simona; Brocca, Paola; Del Favero, Elena; Rondelli, Valeria; Cantù, Laura; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2013-02-01

    Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

  7. DNA probe for detection of the Leptospira interrogans serovar hardjo genotype hardjo-bovis.

    PubMed Central

    LeFebvre, R B

    1987-01-01

    A DNA probe is described for the diagnostic and taxonomic identification of the North American cattle pathogen Leptospira interrogans genotype hardjo-bovis. The probe is specific for this genotype and does not hybridize to genomic DNA of any other leptospire pathogen commonly found in North America. Images PMID:2826538

  8. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  9. Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

    PubMed Central

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2008-01-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

  10. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  11. Imaging of DNA and Protein–DNA Complexes with Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein–DNA complexes, including recent advances in the visualization of protein–DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein–DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein–DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described. PMID:27278886

  12. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    PubMed

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  13. Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Baran, Irina; Ionescu, Diana; Privitera, Simona; Scordino, Agata; Mocanu, Maria Magdalena; Musumeci, Francesco; Grasso, Rosaria; Gulino, Marisa; Iftime, Adrian; Tofolean, Ioana Teodora; Garaiman, Alexandru; Goicea, Alexandru; Irimia, Ruxandra; Dimancea, Alexandru; Ganea, Constanta

    2013-12-01

    The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.

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

    PubMed Central

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Elham

    2015-01-01

    Introduction The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Material and methods Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. Results There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. Conclusions The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes. PMID:26855801

  15. DNA binding and recognition by binuclear transition metal complexes

    NASA Astrophysics Data System (ADS)

    Liu, Changlin; Yan, Rui; Xu, Yan; Yu, Siwang; Liao, Zhanru; Li, Dongfeng; Xu, Hui-Bie F.

    2001-09-01

    The development of small molecules that can bind and recognize DNA with sequence- or stereo-specificity under physiological conditions has been attracting a great interest in chemistry and biochemistry. Here, spectroscopic characterization and gel electrophoresis methods have been utilized to investigate the DNA binding and recognition by a variety of binuclear transition metal complexes. The result indicate that the structures and charges of binuclear transition metal complexes, compositions of coordination spheres, central metal ions and their coordination unsaturation, and separations between two central metal atoms can exert significant effects on the DNA binding and recognition. If there are not intercalative ligands into DNA base pairs or kinetically substitutable ligands by DNA phosphate groups within coordination sphere, the coordination saturation and compact binuclear transition metal complexes weaker bind to DNA than the coordination unsaturation and extended ones to DNA. Since the different transtiometal ions exhibit different affinities to DNA phosphate oxygen atoms, the binding interactions between their binuclear complexes and DNA are controlled by the affinity. He binuclear complexes with one or more negative charges lead to a consequence that they can not efficient associate with DNA, because DNA phosphodiester backbone is negatively charged. Whenthe separations between two central transition metal atoms is more than the distance between two DNA base pairs, the binuclear complexes could bind and recognize the DNA sequence with two or more base pairs. The protonated and positively charged ligands can strengthen the DNA binding and recognition by these binuclear metal complexes. Based on such DNA binding and recognition principles, the binuclear zinc complex designed in the study preferentially bind and recognize the following DNA sequence on pBR322 DNA with binding constant K.

  16. Value of a DNA probe assay (Gen-Probe) compared with that of culture for diagnosis of gonococcal infection.

    PubMed Central

    Vlaspolder, F; Mutsaers, J A; Blog, F; Notowicz, A

    1993-01-01

    The Gen-Probe PACE 2 system for Neisseria gonorrhoeae (GP), which uses a chemiluminescently labeled DNA probe, was compared with conventional culture as the method of reference. A total of 1,750 specimens were collected from 496 females and 623 males visiting the outpatient clinic of the Sexually Transmitted Diseases Department of the Westeinde Hospital, The Hague, The Netherlands, during the year 1991. The prevalences of gonorrhea culture-positive men and women were 14.9 and 7.7%, respectively. The overall positive rate was 8.7%. Sensitivity, specificity, and positive and negative predictive values of GP were 97.1, 99.1, 90.6, and 99.8%, respectively. A total of 12 of 13 patients with positive GP results and negative cultures may have had a gonococcal infection, a conclusion based on clinical symptoms, positive methylene blue smears, and high relative light unit ratios. The DNA probe test can be useful as a suitable screening and diagnostic test for gonorrheal infection in men and women. An advantage of using this DNA probe technique is that simultaneous testing for Chlamydia trachomatis of the same specimen is possible. We also examined whether (all) rRNA had disappeared after adequate treatment for gonococcal and/or chlamydial infection in 30 patients. None of those positive patients showed a positive result in the DNA probe assay after treatment. PMID:8417014

  17. MHF complex senses branched DNA via binding a pair of crossover DNA duplexes

    PubMed Central

    Zhao, Qi; Saro, Dorina; Sachpatzidis, Aristidis; Singh, Thiyam Ramsing; Schlingman, Daniel; Zheng, Xiao-Feng; Mack, Andrew; Tsai, Miaw-Sheue; Mochrie, Simon; Regan, Lynne; Meetei, Amom Ruhikanta; Sung, Patrick; Xiong, Yong

    2014-01-01

    The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anemia (FA) pathway of DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the FA DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA binding mode of MHF. The structure suggests an MHF preference for branched DNA over double stranded DNA through engaging two duplex arms, which is supported by single molecule studies. Biochemical analyses verify that MHF preferentially engage DNA forks or various four-way junctions independent of the junction-site structure. Genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results provide insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance. PMID:24390579

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

    SciTech Connect

    Hecht, N.B.

    1987-10-01

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

  19. DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1.

    PubMed

    Grabowska, Iwona; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Olejniczak, Agnieszka B; Leśnikowski, Zbigniew J; Radecki, Jerzy; Radecka, Hanna

    2014-01-15

    In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region. PMID:23958581

  20. Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

    PubMed

    Anderson, Brooke A; Hrdlicka, Patrick J

    2016-04-15

    The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and-more recently-engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics. PMID:26998918

  1. Probing Evolutionary Patterns in Neotropical Birds through DNA Barcodes

    PubMed Central

    Kerr, Kevin C. R.; Lijtmaer, Darío A.; Barreira, Ana S.; Hebert, Paul D. N.; Tubaro, Pablo L.

    2009-01-01

    Background The Neotropical avifauna is more diverse than that of any other biogeographic region, but our understanding of patterns of regional divergence is limited. Critical examination of this issue is currently constrained by the limited genetic information available. This study begins to address this gap by assembling a library of mitochondrial COI sequences, or DNA barcodes, for Argentinian birds and comparing their patterns of genetic diversity to those of North American birds. Methodology and Principal Findings Five hundred Argentinian species were examined, making this the first major examination of DNA barcodes for South American birds. Our results indicate that most southern Neotropical bird species show deep sequence divergence from their nearest-neighbour, corroborating that the high diversity of this fauna is not based on an elevated incidence of young species radiations. Although species ages appear similar in temperate North and South American avifaunas, patterns of regional divergence are more complex in the Neotropics, suggesting that the high diversity of the Neotropical avifauna has been fueled by greater opportunities for regional divergence. Deep genetic splits were observed in at least 21 species, though distribution patterns of these lineages were variable. The lack of shared polymorphisms in species, even in species with less than 0.5M years of reproductive isolation, further suggests that selective sweeps could regularly excise ancestral mitochondrial polymorphisms. Conclusions These findings confirm the efficacy of species delimitation in birds via DNA barcodes, even when tested on a global scale. Further, they demonstrate how large libraries of a standardized gene region provide insight into evolutionary processes. PMID:19194495

  2. Immunodetection of human topoisomerase I-DNA covalent complexes.

    PubMed

    Patel, Anand G; Flatten, Karen S; Peterson, Kevin L; Beito, Thomas G; Schneider, Paula A; Perkins, Angela L; Harki, Daniel A; Kaufmann, Scott H

    2016-04-01

    A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitorsin vitroandin vivo. PMID:26917015

  3. Immunodetection of human topoisomerase I-DNA covalent complexes

    PubMed Central

    Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

    2016-01-01

    A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo. PMID:26917015

  4. Detection of specific DNA sequences with short biotin-labeled probes.

    PubMed

    Chu, B C; Orgel, L E

    1985-08-01

    We have developed a simple, general synthesis of nonradioactive DNA probes in which biotin is attached to the 5'-terminal phosphate of an oligodeoxyribonucleotide 16 bases long via an ethylenediamine or hexamethylenediamine linker. The products are stable under normal hybridization conditions. They hybridize to target DNA as efficiently as the underivatized oligodeoxyribonucleotide. Color development, using a commercially available kit, is complete within 3 hr using the biotin-detection method. The sensitivity of detection of homologous DNA with a probe to which biotin was attached via a hexamethylenediamine linker is about one-tenth of that achieved overnight by autoradiography with the corresponding 32P-labeled probe. PMID:4042814

  5. Probing DNA Helicase Kinetics with Temperature‐Controlled Magnetic Tweezers

    PubMed Central

    Gollnick, Benjamin; Carrasco, Carolina; Zuttion, Francesca; Gilhooly, Neville S.; Dillingham, Mark S.

    2015-01-01

    Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single‐molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface‐coupled microscopy technique, such as tethered particle motion (TPM), nanopore‐based sensing of biomolecules, or super‐resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double‐stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase‐nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub‐saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21–24 k B T) are obtained by comparing results from MT and stopped‐flow bulk assays. Single‐molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature‐controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano‐biotechnology. PMID:25400244

  6. Fluorescent silver nanocluster DNA probes for multiplexed detection using microfluidic capillary electrophoresis.

    PubMed

    Del Bonis-O'Donnell, Jackson Travis; Fygenson, Deborah K; Pennathur, Sumita

    2015-03-01

    DNA-stabilized fluorescent silver nanoclusters (AgNC DNA) are a new class of fluorophore that are formed by sequence specific interactions between silver and single-stranded DNA. By incorporating both target-binding and fluorescent-reporting sequences into a single synthetic DNA oligomer, AgNC DNA probes eliminate the need to conjugate dye or quencher molecules. In this study, we modify a AgNC DNA probe to demonstrate single-color multiplexed detection of DNA targets. We show that appending different lengths of poly-dT to the probe sequences tunes the electrophoretic mobility of AgNC DNA probes without affecting their fluorescence spectra. We use this to introduce a set of AgNC DNA probes selective for Hepatitis A, B and C target sequences that can be processed together in a simple, single-step protocol and distinguished with a resolution of 3.47 and signal to noise ratio of 17.23 in under 10 seconds by microfluidic capillary electrophoresis. PMID:25601044

  7. A Microfluidic Microbeads Fluorescence Assay with Quantum Dots-Bead-DNA Probe.

    PubMed

    Ankireddy, S R; Kim, Jongsung

    2016-03-01

    A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA. PMID:27455729

  8. Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes

    NASA Astrophysics Data System (ADS)

    Shah, Pratik; Cho, Seok Keun; Waaben Thulstrup, Peter; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

    2014-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ˜21 nt to ˜25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

  9. Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes.

    PubMed

    Shah, Pratik; Cho, Seok Keun; Thulstrup, Peter Waaben; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

    2014-01-31

    MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded. PMID:24393838

  10. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes

    PubMed Central

    Storhoff, James J; Lucas, Adam D; Garimella, Viswanadham; Bao, Y Paul; Müller, Uwe R

    2005-01-01

    Nucleic acid diagnostics is dominated by fluorescence-based assays that use complex and expensive enzyme-based target or signal-amplification procedures1–6. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. We have developed a ‘spot-and-read’ colorimetric detection method for identifying nucleic acid sequences based on the distance-dependent optical properties of gold nanoparticles. In this assay, nucleic acid targets are recognized by DNA-modified gold probes, which undergo a color change that is visually detectable when the solutions are spotted onto an illuminated glass waveguide. This scatter-based method enables detection of zeptomole quantities of nucleic acid targets without target or signal amplification when coupled to an improved hybridization method that facilitates probe-target binding in a homogeneous format. In comparison to a previously reported absorbance-based method7, this method increases detection sensitivity by over four orders of magnitude. We have applied this method to the rapid detection of mecA in methicillin-resistant Staphylococcus aureus genomic DNA samples. PMID:15170215

  11. A versatile proximity-dependent probe based on light-up DNA-scaffolded silver nanoclusters.

    PubMed

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

    2016-02-01

    It is well-known that proximity-dependent probes containing an analyte recognization site and a signal formation domain could be assembled specifically into a sandwich-like structure (probe-analyte-probe) via introducing an analyte. In this work, using the design for zirconium ion (Zr(4+)) detection as the model, we develop a novel and reliable proximity-dependent DNA-scaffolded silver nanocluster (DNA/AgNC) probe for Zr(4+) detection via target-induced emitter proximity. The proposed strategy undergoes the two following processes: target-mediated emitter pair proximity as target recognition implement and the synthesis of DNA/AgNCs with fluorescence as a signal reporter. Upon combination of the rationally designed probe with Zr(4+), the intact templates were obtained according to the -PO3(2-)-Zr(4+)-PO3(2-)- pattern. The resultant structure with an emitter pair serves as a potent template to achieve highly fluorescent DNA/AgNCs. To verify the universality of the proposed proximity-dependent DNA/AgNC probe, we extend the application of the proximity-dependent probe to DNA and adenosine triphosphate (ATP) detection by virtue of a specific DNA complementary sequence and ATP aptamer as a recognition unit, respectively. The produced fluorescence enhancement of the DNA/AgNCs in response to the analyte concentration allows a quantitative evaluation of the target, including Zr(4+), DNA, and ATP with detection limits of ∼3.00 μM, ∼9.83 nM, and ∼0.81 mM, respectively. The proposed probe possesses good performance with simple operation, cost-effectiveness, good selectivity, and without separation procedures. PMID:26814697

  12. Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

    PubMed Central

    Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

    2009-01-01

    Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

  13. Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe.

    PubMed

    Zhao, Haiyan; Wang, Lei; Zhu, Jing; Wei, Haiping; Jiang, Wei

    2015-06-01

    Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10(-10) mol L(-1) HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples. PMID:25863386

  14. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  15. Hairpin DNA probes based on target-induced in situ generation of luminescent silver nanoclusters.

    PubMed

    Xiao, Yan; Wu, Zhengjun; Wong, Kwok-Yin; Liu, Zhihong

    2014-05-14

    Novel hairpin DNA probes are designed and constructed based on target-induced in situ generation of luminescent silver nanoclusters. This design allows specific and versatile detection of diverse targets with easy operation and low cost. PMID:24686790

  16. A luminescent beta-cyclodextrin-based Ru(phen)3 complex as DNA compactor, enzyme inhibitor, and translocation tracer.

    PubMed

    Liu, Yu; Chen, Yong; Duan, Zhong-Yu; Feng, Xi-Zeng; Hou, Sen; Wang, Chen; Wang, Rui

    2007-11-01

    A beta-cyclodextrin-based Ru(phen)(3) complex (1) has been synthesized and exhibits good luminescent behavior. Atomic force microscopic and scanning electron microscopic studies show that 1 can induce the aggregation of originally circular DNA to toroidal or spherical shapes. The morphology of these DNA aggregates changes following a pathway of naked circular DNA --> toroid with gaps --> solid toroid --> spherical aggregate, depending on the different 1/DNA (w/w) ratios, and their average diameters vary from the nanometer to micrometer scale. Owing to its capability of inducing the aggregation of DNA, 1 can be used as an inhibitor for DNA topoisomerase and DNA cleavage enzymes. Further studies by means of fluorescence microscopy indicate that 1 can also efficiently trace the translocation of DNA into 293T cells (the human embryonic kidney cell line). These observations consequently establish 1 as not only a potential DNA carrier but also a fluorescent DNA probe. PMID:19206682

  17. Spectroscopic studies on the interaction between anthragallol and DNA using of ethidium bromide as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Gao, Yan; Li, Junsheng; Huang, Guoxia; Yan, Liujuan; Dong, Zhen

    2015-04-01

    The interaction of DNA with anthragallol (Ant) was investigated using ethidium bromide (EB) as a fluorescence probe, and the binding mechanism of Ant with DNA was researched via viscosity measurements. The results indicate that there is a complex of Ant and DNA, as confirmed by Ultraviolet visible absorption spectroscopy (UV-vis), Fluorescent and Resonance Light Scattering spectrum (RLS) and viscosity measurements. Ant molecules could intercalate with the base pairs of DNA as evidenced by the hyperchromic effect of absorption spectra, the relative viscosity of DNA and significant increases in the melting temperature. The binding constants of Ant and DNA were obtained by the fluorescence quenching technique. Furthermore, the binding mechanisms of the reaction of Ant with DNA were also investigated. The RLS assay successfully evaluated the saturated value and measured the potential toxicity of Ant. Adriamycin, chrysophanol, rhein, and alizarin can be used as references to build a method based on the mechanism of interactions with DNA and the DNA-saturation binding value to rapidly evaluate the potential toxicity of Ant.

  18. DNA probes for identification of enteroinvasive Escherichia coli.

    PubMed Central

    Gomes, T A; Toledo, M R; Trabulsi, L R; Wood, P K; Morris, J G

    1987-01-01

    Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55). All 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes. Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile. Motile strains of serotype O144:H25 were for the first time characterized as invasive by hybridization with the probes. PMID:3312292

  19. Polyamide fluorescent probes for visualization of repeated DNA sequences in living cells.

    PubMed

    Nozeret, Karine; Loll, François; Escudé, Christophe; Boutorine, Alexandre S

    2015-03-01

    DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells. PMID:25639955

  20. A reagentless DNA-based electrochemical silver(I) sensor for real time detection of Ag(I) - the effect of probe sequence and orientation on sensor response.

    PubMed

    Wu, Yao; Lai, Rebecca Y

    2016-06-01

    Ag(I) is known to interact with cytosine (C) via the formation C-Ag(I)-C complexes. The authors have utilized this concept to design six electrochemical Ag(I) sensors using C-rich DNA probes. Alternating current voltammetry and cyclic voltammetry were used to analyze the sensors. The results show that the dual-probe sensors that require the use of both 5'- and 3'-thiolated DNA probes are not suitable for this application, the differences in probe orientation impedes formation of C-Ag(I)-C complexes. Sensors fabricated with DNA probes containing both thymine (T) and C, independent of the location of the alkanethiol linker, do not response to Ag(I) either; T-T mismatches destabilize the duplex even in the presence of Ag(I). However, sensors fabricated with DNA probes containing both adenine (A) and C are ideal for this application, owing to the formation of C-Ag(I)-C complexes, as well as other lesser known interactions between A and Ag(I). Both sensors are sensitive, specific and selective enough to be used in 50% human saliva. They can also be used to detect silver sulfadiazine, a commonly prescribed antimicrobial drug. With further optimization, this sensing strategy may offer a promising approach for detection of Ag(I) in environmental and clinical samples. PMID:26901685

  1. In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.

    PubMed

    Shah, Pratik; Thulstrup, Peter Waaben; Cho, Seok Keun; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Bjerrum, Morten Jannik; Yang, Seong Wook

    2014-05-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method. PMID:24616905

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

    PubMed

    Kavanagh, Paul; Leech, Dónal

    2006-04-15

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

  3. Probing Resonances of the Dirac Equation with Complex Momentum Representation

    NASA Astrophysics Data System (ADS)

    Li, Niu; Shi, Min; Guo, Jian-You; Niu, Zhong-Ming; Liang, Haozhao

    2016-08-01

    Resonance plays critical roles in the formation of many physical phenomena, and several methods have been developed for the exploration of resonance. In this work, we propose a new scheme for resonance by solving the Dirac equation in the complex momentum representation, in which the resonant states are exposed clearly in the complex momentum plane and the resonance parameters can be determined precisely without imposing unphysical parameters. Combined with the relativistic mean-field theory, this method is applied to probe the resonances in 120120 with the energies, widths, and wave functions being obtained. Compared to other methods, this method is not only very effective for narrow resonances, but also can be reliably applied to broad resonances.

  4. Probing Resonances of the Dirac Equation with Complex Momentum Representation.

    PubMed

    Li, Niu; Shi, Min; Guo, Jian-You; Niu, Zhong-Ming; Liang, Haozhao

    2016-08-01

    Resonance plays critical roles in the formation of many physical phenomena, and several methods have been developed for the exploration of resonance. In this work, we propose a new scheme for resonance by solving the Dirac equation in the complex momentum representation, in which the resonant states are exposed clearly in the complex momentum plane and the resonance parameters can be determined precisely without imposing unphysical parameters. Combined with the relativistic mean-field theory, this method is applied to probe the resonances in ^{120}Sn with the energies, widths, and wave functions being obtained. Compared to other methods, this method is not only very effective for narrow resonances, but also can be reliably applied to broad resonances. PMID:27541464

  5. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  7. A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe.

    PubMed

    Xing, Xiao-Jing; Xiao, Wan-Lu; Liu, Xue-Guo; Zhou, Ying; Pang, Dai-Wen; Tang, Hong-Wu

    2016-04-15

    We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies. PMID:26655184

  8. Luminescent chiral lanthanide(III) complexes as potential molecular probes

    PubMed Central

    Muller, Gilles

    2009-01-01

    This perspective gives an introduction into the design of luminescent lanthanide(III)-containing complexes possessing chiral properties and used to probe biological materials. The first part briefly describes general principles, focusing on the optical aspect (i.e. lanthanide luminescence, sensitization processes) of the most emissive trivalent lanthanide ions, europium and terbium, incorporated into molecular luminescent edifices. This is followed by a short discussion on the importance of chirality in the biological and pharmaceutical fields. The second part is devoted to the assessment of the chiroptical spectroscopic tools available (typically circular dichroism and circularly polarized luminescence) and the strategies used to introduce a chiral feature into luminescent lanthanide(III) complexes (chiral structure resulting from a chiral arrangement of the ligand molecules surrounding the luminescent center or presence of chiral centers in the ligand molecules). Finally, the last part illustrates these fundamental principles with recent selected examples of such chiral luminescent lanthanide-based compounds used as potential probes of biomolecular substrates. PMID:19885510

  9. Structure of Escherichia coli AlkA in Complex with Undamaged DNA

    SciTech Connect

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; Verdine, Gregory L

    2010-11-22

    Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures with that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions.

  10. Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid.

    PubMed

    Sulaiman, Saba A J; Al-Rasbi, Ghalia S; Abou-Zied, Osama K

    2016-05-01

    Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein-ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein-binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. PMID:26910188

  11. Iridium Complexes as a Roadblock for DNA Polymerase during Amplification.

    PubMed

    Chandra, Falguni; Kumar, Prashant; Tripathi, Suman Kumar; Patra, Srikanta; Koner, Apurba L

    2016-07-01

    Iridium-based metal complexes containing polypyridyl-pyrazine ligands show properties of DNA intercalation. They serve as roadblocks to DNA polymerase activity, thereby inhibiting the polymerization process. Upon the addition of increasing concentrations of these iridium complexes, a rapid polymerase chain reaction (PCR)-based assay reveals the selective inhibition of the DNA polymerization process. This label-free approach to study the inhibition of fundamental cellular processes via physical roadblock can offer an alternative route toward cancer therapy. PMID:27240728

  12. An overview of the structures of protein-DNA complexes

    PubMed Central

    Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

    2000-01-01

    On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

  13. DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection.

    PubMed

    Hao, Rong-Zhang; Song, Hong-Bin; Zuo, Guo-Min; Yang, Rui-Fu; Wei, Hong-Ping; Wang, Dian-Bing; Cui, Zong-Qiang; Zhang, ZhiPing; Cheng, Zhen-Xing; Zhang, Xian-En

    2011-04-15

    The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis. PMID:21315574

  14. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    PubMed

    Paquet, Françoise; Delalande, Olivier; Goffinont, Stephane; Culard, Françoise; Loth, Karine; Asseline, Ulysse; Castaing, Bertrand; Landon, Celine

    2014-01-01

    In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA. PMID:24558431

  15. New reagents for the introduction of reactive functional groups into chemically synthesized DNA probes.

    PubMed

    Skrzypczynski, Zbigniew; Wayland, Sarah

    2003-01-01

    An efficient and versatile preparative approach is described, allowing for the preparation of DNA probes modified with an aldehyde group at the 3'- or 5'-end. The developed synthetic strategy allows for the preparation of a new family of phosphoramidites and solid supports compatible with the automated synthesis of modified oligonucleotide probes. These new reagents were prepared from intermediates 3 and 3a, obtained from the commercially available aleuritic acid 1. It was demonstrated that the new phosphoramidite reagents also could be used as new types of cleavable linkers. A new and efficient method for the production of 5' aldehyde-labeled DNA probes was developed. PMID:12757390

  16. Structure and optoelectrical properties of photopolymerized PAn/DNA complex

    NASA Astrophysics Data System (ADS)

    Kobayashi, Norihisa; Morimoto, Taro; Ushikubo, Takahiro

    2007-09-01

    A Polyaniline (PAn)/ DNA complex has been successfully prepared by the photopolymerization of dimeric aniline via photocatalytic reaction of Ru(bpy) 3 2+ in the presence of DNA. The reaction occurs even in the solution at pH 3.0 - 6.0, due to the specific local "lower-pH" environment provided by DNA. The PAn in the complex has ordered structure associated with double-helical DNA. The complex contains photocatalyst, Ru(bpy) 3 2+, even after purification and the Ru(bpy) 3 2+ also works as emitting material. A Ru(bpy) 3 2+ complex-based red-emitting diode with a fast turn-on response was successfully fabricated by employing this novel, processable and water-soluble PAn/DNA complex.

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

    SciTech Connect

    Dittmer, Joy

    1996-05-01

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

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

    SciTech Connect

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

    2011-03-08

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

  19. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  20. Electrochemical probe for the monitoring of DNA-protein interactions.

    PubMed

    Meunier-Prest, Rita; Bouyon, Alice; Rampazzi, Eve; Raveau, Suzanne; Andreoletti, Pierre; Cherkaoui-Malki, Mustapha

    2010-08-15

    Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower than the calculated K(D) for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K(D) from 0.46 microM to more than 25 microM. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range. PMID:20447818

  1. Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells.

    PubMed

    Kwasniewska, Jolanta; Grabowska, Marta; Kwasniewski, Miroslaw; Kolano, Bozena

    2012-06-01

    We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment. PMID:22556029

  2. 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

  3. Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients’ acute phase serum & CSF samples

    PubMed Central

    Kumar, Jyoti S.; Parida, Manmohan; Lakshmana Rao, P.V.

    2013-01-01

    Background & objectives: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. Methods: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. Results: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O’Nyong Nyong, Semlinki forest and Sindbis viruses. Interpretation & conclusion: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose. PMID:24056565

  4. Time-resolved probes based on guanine/thymine-rich DNA-sensitized luminescence of terbium(III).

    PubMed

    Zhang, Min; Le, Huynh-Nhu; Jiang, Xiao-Qin; Yin, Bin-Cheng; Ye, Bang-Ce

    2013-12-01

    In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb(3+)) using a designed guanine/thymine-rich DNA (5'-[G3T]5-3') as an antenna ligand, in which [G3T]5 improved the luminescence of Tb(3+) by 3 orders of magnitude due to energy transfer from nucleic acids to Tb(3+) (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag(+), and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb(3+) (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg(2+) can efficiently quench the luminescence of Tb(3+) sensitized by [G3T]5 (Tb(3+)/[G3T]5); however, biothiols are readily applicable to selectively grab Hg(2+) for restoration of the luminescence of Tb(3+)/[G3T]5 initially quenched by Hg(2+), which can be used for "turn on" detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb(3+). However, in the presence of Ag(+), Ag(+) can combine the C base of c[G3T]5 to form C-Ag(+)-C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb(3+). Therefore, the Tb(3+)/[G3T]5/c[G3T]5 probe can be applied to detect Ag(+) in a "turn on" format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb(3+), producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb(3+). Results and methods reported here suggest that a guanine/thymine-rich DNA

  5. Self-quenching DNA probes based on aggregation of fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Schafer, Gabriela; Muller, Matthias; Hafner, Bernhard; Habl, Gregor; Nolte, Oliver; Marme, Nicole; Knemeyer, Jens-Peter

    2005-04-01

    Here we present a novel class of self-quenching, double-labeled DNA probes based on the formation of non fluorescent H-type dye dimers. We therefore investigated the aggregation behavior of the red-absorbing oxazine derivative MR121 and found a dimerization constant of about 3000 M-1. This dye was successfully used to develop hairpin-structured as well as linear self-quenching DNA probes that report the presence of the target DNA by an increase of the fluorescence intensity by a factor of 3 to 12. Generally fluorescence quenching of the hairpin-structure probes is more efficient compared to the linear probes, whereas the kinetic of the fluorescence increase is significantly slower. The new probes were used for the identification of different mycobacteria and their antibiotic resistant species. As a test system a probe for the identification of a DNA sequence specific for the Mycobacterium xenopi was synthesized differing from the sequence of the Mycobacterium fortuitum by 6 nucleotides. Furthermore we developed a method for the discrimination between the sequences of the wild type and an antibiotic resistant species of Mycobacterium tuberculosis. Both sequences differ by just 2 nucleotides and were detected specifically by the use of competing olignonucleotides.

  6. New hairpin-structured DNA probes: alternatives to classical molecular beacons

    NASA Astrophysics Data System (ADS)

    Friedrich, Achim; Habl, Gregor; Sauer, Markus; Wolfrum, Jürgen; Hoheisel, Jörg; Marmé, Nicole; Knemeyer, Jens-Peter

    2007-02-01

    In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

  7. Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes.

    PubMed Central

    Wong, D L; Pavlovich, J G; Reich, N O

    1998-01-01

    We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. Eco RI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies. PMID:9421528

  8. Effective delivery of DNA into tumor cells and tissues by electroporation of polymer-DNA complex.

    PubMed

    Kang, Jeong-Hun; Toita, Riki; Niidome, Takuro; Katayama, Yoshiki

    2008-07-01

    Electroporation is a useful means for non-viral gene delivery. Here, we investigated the use of electroporation to deliver polymer-DNA complexes into living cells using a protein kinase C (PKC)alpha-responsive polymer. The polymer was complexed with a luciferase-encoding DNA and electroporated into B16 melanoma cells. Gene expression from polymer-DNA complexes was 3- to 5-fold higher than from naked DNA. Moreover, after introduction of the polymer-DNA complex into tissues, luciferase levels were >2-fold higher in B16 melanoma tumors than in normal skin tissue. These results suggest that the combination of our polymer and electroporation is useful for the effective delivery of DNA into tumors. PMID:18375054

  9. 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

  10. Probing neutral atmospheric collision complexes with anion photoelectron imaging

    NASA Astrophysics Data System (ADS)

    Jarrold, Caroline

    Photodetachment of anionic precursors of neutral collision complexes offers a way to probe the effects of symmetry-breaking collision events on the electronic structure of normally transparent molecules. We have measured the anion photoelectron imaging (PEI) spectra of a series of O2- X complexes, where X is a volatile organic molecule with atmospheric relevance, to determine how the electronic properties of various X molecules affect the low-lying electronic structure of neutral O2 undergoing O2 - X collisons. The study was motivated by the catalog of vibrational and electronic absorption lines induced by O2 - O2, O2 - N2, and other collisions. The energies of electronic features observed in the anion PEI spectra of O2- X (X = hexane, hexene, isoprene and benzene) relative to O2- PEI spectroscopic features indicate that photodetachment of the anion does indeed access a repulsive part of the O2 - X potential. In addition, the spectra of the various complexes show an interesting variation in the intensities of transitions to the excited O2(1Δg) . X and O2(1Σg+) . X states relative to the ground O2(3Σg-) . X state. With X = non-polar species such as hexane, the relative intensities of transitions to the triplet and singlet states of O2 . X are very similar to those of isolated O2, while the relative intensity of the singlet band decreases and becomes lower in energy relative to the triplet band for X = polar molecules. A significant enhancement in the intensities of the singlet bands is observed for complexes with X = isoprene and benzene, both of which have low-lying triplet states. The role of the triplet states in isoprene and benzene, and the implications for induced electronic absorption in O2 undergoing collisions with these molecules, are explored. National Science Foundation NSF CHE 1265991.

  11. 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

  12. Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

    PubMed

    Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

    2014-05-01

    DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases. PMID:24497635

  13. Probing the Conformational Distributions of Sub-Persistence Length DNA

    SciTech Connect

    Mastroianni, Alexander; Sivak, David; Geissler, Phillip; Alivisatos, Paul

    2009-06-08

    We have measured the bending elasticity of short double-stranded DNA (dsDNA) chains through small-angle X-ray scattering from solutions of dsDNA-linked dimers of gold nanoparticles. This method, which does not require exertion of external forces or binding to a substrate, reports on the equilibrium distribution of bending fluctuations, not just an average value (as in ensemble FRET) or an extreme value (as in cyclization), and in principle provides a more robust data set for assessing the suitability of theoretical models. Our experimental results for dsDNA comprising 42-94 basepairs (bp) are consistent with a simple worm-like chain model of dsDNA elasticity, whose behavior we have determined from Monte Carlo simulations that explicitly represent nanoparticles and their alkane tethers. A persistence length of 50 nm (150 bp) gave a favorable comparison, consistent with the results of single-molecule force-extension experiments on much longer dsDNA chains, but in contrast to recent suggestions of enhanced flexibility at these length scales.

  14. Photoinduced interactions of two dirhodium complexes with d(GTCGAC)2 probed by 2D NOESY

    PubMed Central

    Palmer, Alycia M.; Knoll, Jessica D.; Turro, Claudia

    2015-01-01

    The interactions between the 6-mer duplex oligonucleotide d(GTCGAC)2 and the photoactive dirhodium complexes cis-H,H-[Rh2(HNOCCH3)2(L)(CH3CN)4]2+, where L represents bpy (1, 2,2´-bipyridine) and dppz (2, dipyrido[3,2-a:2′,3′-c]phenazine), were probed using 2D 1H–1H NOESY NMR spectroscopy. Complex 1 does not interact with the duplex in the dark, but binds covalently to the terminal guanine following irradiation with visible light. Similar behavior was observed for 2, but in addition to the photoinduced covalent DNA binding, the planar dppz ligand of the complex shields the terminal cytosine protons after irradiation. The results are consistent with photoinduced guanine coordination and end-capping of the duplex through π-stacking interactions with the terminal GC base pair. These data show that in the presence of the 6-mer duplex oligonucleotide, 1 and 2 exhibit photoinduced covalent binding to DNA. In addition, the π-stacking interactions of 2 with the duplex are enhanced upon irradiation. PMID:25557067

  15. Real-time observation of DNA repair: 2-aminopurine as a molecular probe

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Butcher, Christina E.; Oh, Dennis H.

    2008-02-01

    Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched. However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of DNA repair in both populations of cells as well as single cells.

  16. Immobilization of human papillomavirus DNA probe for surface plasmon resonance imaging

    NASA Astrophysics Data System (ADS)

    Chong, Xinyuan; Ji, Yanhong; Ma, Suihua; Liu, Le; Liu, Zhiyi; Li, Yao; He, Yonghong; Guo, Jihua

    2009-08-01

    Human papillomavirus (HPV) is a kind of double-stranded DNA virus whose subspecies have diversity. Near 40 kinds of subspecies can invade reproductive organ and cause some high risk disease, such as cervical carcinoma. In order to detect the type of the subspecies of the HPV DNA, we used the parallel scan spectral surface plasmon resonance (SPR) imaging technique, which is a novel type of two- dimensional bio-sensing method based on surface plasmon resonance and is proposed in our previous work, to study the immobilization of the HPV DNA probes on the gold film. In the experiment, four kinds of the subspecies of the HPV DNA (HPV16, HPV18, HPV31, HPV58) probes are fixed on one gold film, and incubate in the constant temperature condition to get a HPV DNA probe microarray. We use the parallel scan spectral SPR imaging system to detect the reflective indices of the HPV DNA subspecies probes. The benefits of this new approach are high sensitive, label-free, strong specificity and high through-put.

  17. Site-Specific DNA Structural and Dynamic Features Revealed by Nucleotide-Independent Nitroxide Probes

    SciTech Connect

    Popova, Anna; Kalai, Tamas; Hideg, Kalman; Qin, Peter Z.

    2009-09-15

    In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label (e.g., duplex center vs termini) and the surrounding DNA sequence. This indicates that R5 and R5a can provide information on the DNA local environment at the level of an individual nucleotide. As these probes can be attached to arbitrary nucleotides within a nucleic acid sequence, they may provide a means to “scan” a given DNA molecule in order to interrogate its local structural and dynamic features.

  18. Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence.

    PubMed Central

    Erhardt, A; Schaefer, S; Athanassiou, N; Kann, M; Gerlich, W H

    1996-01-01

    We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test. PMID:8818875

  19. Proceedings of "Optical Probes of Dynamics in Complex Environments"

    SciTech Connect

    Sension, R; Tokmakoff, A

    2008-04-01

    This document contains the proceedings from the symposium on Optical Probes of Dynamics in Complex Environments, which organized as part of the 235th National Meeting of the American Chemical Society in New Orleans, LA from April 6 to 10, 2008. The study of molecular dynamics in chemical reaction and biological processes using time ƒresolved spectroscopy plays an important role in our understanding of energy conversion, storage, and utilization problems. Fundamental studies of chemical reactivity, molecular rearrangements, and charge transport are broadly supported by the DOE Office of Science because of their role in the development of alternative energy sources, the understanding of biological energy conversion processes, the efficient utilization of existing energy resources, and the mitigation of reactive intermediates in radiation chemistry. In addition, time resolved spectroscopy is central to all of DOEs grand challenges for fundamental energy science. This symposium brought together leaders in the field of ultrafast spectroscopy, including experimentalists, theoretical chemists, and simulators, to discuss the most recent scientific and technological advances. DOE support for this conference was used to help young US and international scientists travel to the meeting. The latest technology in ultrafast infrared, optical, and xray spectroscopy and the scientific advances that these methods enable were covered. Particular emphasis was placed on new experimental methods used to probe molecular dynamics in liquids, solids, interfaces, nanostructured materials, and biomolecules.

  20. Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

    SciTech Connect

    Liehr, T.; Weise, A.; Heller, A.; Starke, H.; Mrasek, K.; Kuechler, A.; Weier, H.-U.G.; Claussen, U.

    2003-06-23

    Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few mega base pairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of over lapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wave length intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific micro dissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region specific paints, but do not readily allow positioning of break points on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

  1. 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.

  2. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  3. Molecular dynamics simulations of DNA-polycation complexes

    NASA Astrophysics Data System (ADS)

    Ziebarth, Jesse; Wang, Yongmei

    2008-03-01

    A necessary step in the preparation of DNA for use in gene therapy is the packaging of DNA with a vector that can condense DNA and provide protection from degrading enzymes. Because of the immunoresponses caused by viral vectors, there has been interest in developing synthetic gene therapy vectors, with polycations emerging as promising candidates. Molecular dynamics simulations of the DNA duplex CGCGAATTCGCG in the presence of 20 monomer long sequences of the polycations, poly-L-lysine (PLL) and polyethyleneimine (PEI), with explicit counterions and TIP3P water, are performed to provide insight into the structure and formation of DNA polyplexes. After an initial separation of approximately 50 å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes upon complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, and rarely occupy electronegative sites in either the major or minor grooves. Differences between complexation with PEI and PLL will be discussed.

  4. Probing DNA with micro- and nanocapillaries and optical tweezers

    NASA Astrophysics Data System (ADS)

    Steinbock, L. J.; Otto, O.; Skarstam, D. R.; Jahn, S.; Chimerel, C.; Gornall, J. L.; Keyser, U. F.

    2010-11-01

    We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

  5. Two closely related nickel complexes have different effects on DNA damage and cell viability.

    PubMed

    Matkar, Smita S; Wrischnik, Lisa A; Jones, Patrick R; Hellmann-Blumberg, Utha

    2006-05-12

    Nickel is considered a weak carcinogen. It is known to interact with DNA and DNA-binding proteins. The ability of certain nickel compounds to cleave DNA has been exploited mainly for research purposes and less for developing new anticancer drugs. Here we compare the interactions of two closely related nickel complexes, [NiCR]2+ and [Ni(CR-2H)]2+, with DNA. CR stands for 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1(17),2,11,13,15-pentaene. [NiCR]2+ has been used in the past as a structure-specific probe for RNA and DNA oligonucleotides in the presence of oxidizing agent but little is known about the biological effects of either complex. Our results show that [Ni(CR-2H)]2+ can damage DNA in vivo and in vitro in the absence of an added oxidizing agent and has an IC50 of 70 microM in human breast cancer cells whereas [NiCR]2+ and NiCl2 do not exhibit significant cytotoxicity. However, both [NiCR]2+ and [Ni(CR-2H)]2+ bind to the minor groove of double-stranded DNA. PMID:16563351

  6. Rapid detection of specific genes from human genomic DNA using the microbead-quantum dot complexes in microfluidic chip.

    PubMed

    Yoo, Jeong Ha; Kim, Jong Sung

    2013-08-01

    In the clinic, it is important to prepare for single stranded DNA from genomic DNA to detect the target gene. In this study, we have investigated the detection of single stranded DNA (ssDNA) obtained by ultrasonication of human genomic DNA via fluorescence quenching on microfluidic chip with pillars at the channel to entrap the microbead-QD complexes (MQCs). The QDs with carboxyl group bind to microbeads with amine group by EDC/NHS coupling reaction. The thiolated probe DNA conjugates strongly with the metal ions on the surface of QDs. The MQCs were packed into a chamber on the channel blocked by pillars. ssDNA and TOTO-3 (intercalating dye) were introduced into the microchannel. After hybridization of probe DNA and target DNA, fluorescence quenching was observed at the surface of the MQDs by FRET between QD and TOTO-3. This experiment shows the possibility of rapid detection of genomic DNA from clinical samples via microbead-QD complexs on microfluidic chip. PMID:23882750

  7. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

    We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication. Images PMID:3025630

  8. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    NASA Astrophysics Data System (ADS)

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-12-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

  9. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    PubMed Central

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-01-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event. PMID:26678946

  10. Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Niu, Luming; Shaiu, Wenling; Vesenka, James; Larson, Drena D.; Henderson, Eric

    1993-06-01

    We are developing methods for random and site-specific labeling of individual DNA molecules to facilitate manipulation of fragments excised in the atomic force microscope (AFM) and for localization of specific DNA domains, such as protein binding sites and origins of replication. One successful method was to incorporate biotinylated nucleotides at random internal locations or specifically at the ends of linearized DNA molecules in vitro. Following complex formation with 5 nm diameter streptavidin-gold conjugates, chromatographic purification and passive adsorption of the complexes of mica, the biotinylated domains were easily localized in the AFM by virtue of the distinctive size and shape of the streptavidin-gold complex. In many cases unconjugated streptavidin (i.e., lacking gold) was also observed attached to the biotinylated DNA. A second approach to site-specific labeling of DNA for imaging in the AFM was to react DNA with restriction enzymes having sequence-specific binding properties. Like the unconjugated streptavidin-DNA complexes, these enzyme-DNA complexes were visible without attached colloidal gold. Efforts to image DNA labeled in vivo using bromodeoxyuridine (BrdU) and anti-BrdU antibodies are ongoing.

  11. Validation of probe EFD52 (D17S26) for forensic DNA analysis.

    PubMed

    Nelson, M S; Benzinger, E A; Budzynski, M J; Boodee, M T; Matthews, A; Buel, E; Schwartz, M B; von Beroldingen, C; Wampler, R L; Coons, T M

    1996-07-01

    Validation studies that meet TWGDAM (The Working Group on DNA Analysis Methods) and CAC (California Association of Criminalists) guidelines for RFLP (restriction fragment length polymorphism) analysis were performed with the DNA probe EFD52 (D17S26). These studies demonstrate that the probe EFD52 is suitable for forensic casework. No unexpected DNA banding patterns were obtained from controlled studies examining various tissues, sample consistency over many gels, mixtures of body fluids, various substrates, various contaminants and non-human DNA sources. Of all the animal DNAs tested, only one higher primate yielded a single band to EFD52 hybridization. The sensitivity of EFD52 was shown to be comparable to that of other forensic probes. Population frequency distribution tables were prepared from over 4000 alleles and two-locus studies were conducted on nine forensically useful probes. Black, White, Hispanic and Lumbee Indian populations were found to be in Hardy-Weinberg and linkage equilibrium. Comparisons between victim blood standards and epithelial fractions of mixed strains from sexual assault cases were used to demonstrate the robustness of the EFD52 probe in forensic casework. PMID:8754565

  12. 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

  13. 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

  14. Use of Ti Plasmid DNA Probes for Determining Tumorigenicity of Agrobacterium Strains

    PubMed Central

    Burr, Thomas J.; Norelli, John L.; Katz, Barbara H.; Bishop, Andrew L.

    1990-01-01

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 106 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains. Images PMID:16348218

  15. TaqMan probe array for quantitative detection of DNA targets

    PubMed Central

    Liu, Heping; Wang, Hong; Shi, Zhiyang; Wang, Hua; Yang, Chaoyong; Silke, Spering; Tan, Weihong; Lu, Zuhong

    2006-01-01

    To date real-time quantitative PCR and gene expression microarrays are the methods of choice for quantification of nucleic acids. Herein, we described a unique fluorescence resonance energy transfer-based microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 3′ amino-modified TaqMan probes were designed and immobilized on a glass slide composing a regular microarray pattern, and used as probes in the consecutive PCR carried out on the surface. During the extension step of the PCR, 5′ nuclease activity of DNA polymerase will cleave quencher dyes of the immobilized probe in the presence of nucleic acids targets. The increase of fluorescence intensities generated by the change in physical distance between reporter fluorophore and quencher moiety of the probes were collected by a confocal scanner. Using this new approach we successfully monitored five different pathogenic genomic DNAs and analyzed the dynamic characteristics of fluorescence intensity changes on the TaqMan probe array. The results indicate that the TaqMan probe array on a planar glass slide monitors DNA targets with excellent specificity as well as high sensitivity. This set-up offers the great advantage of real-time quantitative detection of DNA targets in a parallel array format.

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

    SciTech Connect

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

    1990-06-01

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

  17. Using Metal Complex Reduced States to Monitor the Oxidation of DNA

    PubMed Central

    Olmon, Eric D.; Hill, Michael G.; Barton, Jacqueline K.

    2011-01-01

    Metallointercalating photooxidants interact intimately with the base stack of double-stranded DNA and exhibit rich photophysical and electrochemical properties, making them ideal probes for the study of DNA-mediated charge transport (CT). The complexes [Rh(phi)2(bpy′)]3+ (phi = 9,10-phenanthrenequinone diimine; bpy′ = 4-methyl-4′-(butyric acid)-2,2′-bipyridine), [Ir(ppy)2(dppz′)]+ (ppy = 2-phenylpyridine; dppz′ = 6-(dipyrido[3,2-a:2′,3′-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)3(dppz)(py′)]+ (dppz = dipyrido[2,3-a:2′,3′-c]phenazine; py′ = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA in order to compare their photooxidation efficiencies. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that the reduced metal states form following excitation of the conjugates at 355 nm. Electrochemical experiments and kinetic analysis of the TA decays indicate that the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA are the primary factors responsible for the trend observed in the guanine oxidation yield of the three complexes. PMID:22043853

  18. Microarray long oligo probe designing for Escherichia coli: an in-silico DNA marker extraction

    PubMed Central

    Behzadi, Payam; Najafi, Ali; Behzadi, Elham

    2016-01-01

    Introduction Urinary tract infections are predominant diseases which may be caused by different pathogenic microorganisms, particularly Escherichia coli (E.coli). DNA microarray technology is an accurate, rapid, sensitive, and specific diagnostic tool which may lead to definite diagnosis and treatment of several infectious diseases. DNA microarray is a multi-process method in which probe designing plays an important. Therefore, the authors of the present study have tried to design a range of effective and proper long oligo microarray probes for detection and identification of different strains of pathogenic E.coli and in particular, uropathogenic E.coli (UPEC). Material and methods E.coli O26 H11 11368 uid41021 was selected as the standard strain for probe designing. This strain encompasses the largest nucleotide sequence and the most number of genes among other pathogenic strains of E.coli. For performing this in silico survey, NCBI database, GReview Server, PanSeq Server, Oligoanalyzer tool, and AlleleID 7.7 were used to design accurate, appropriate, effective, and flexible long oligo microarray probes. Moreover, the genome of E.coli and its closely related microorganisms were compared. Results In this study, 15 long oligo microarray probes were designed for detecting and identifying different strains of E.coli such as UPEC. These probes possessed the best physico-chemical characteristics. The functional and structural properties of the designed probes were recognized by practical tools and softwares. Conclusions The use of reliable advanced technologies and methodologies for probe designing guarentees the high quality of microarray probes and makes DNA microarray technology more flexible and an effective diagnostic technique. PMID:27123336

  19. Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of Campylobacter spp. in Human Stool Specimens

    PubMed Central

    Collins, Evelyn; Glennon, Maura; Hanley, Shirley; Murray, Anne-Marie; Cormican, Martin; Smith, Terry; Maher, Majella

    2001-01-01

    DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter. PMID:11682549

  20. Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

    PubMed Central

    Dillingham, Mark S.; Tibbles, Katherine L.; Hunter, Jackie L.; Bell, Jason C.; Kowalczykowski, Stephen C.; Webb, Martin R.

    2008-01-01

    The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases. PMID:18599625

  1. Study of concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    NASA Astrophysics Data System (ADS)

    Roshila, M. L.; Hashim, U.; Azizah, N.

    2016-07-01

    This paper mainly illustrates regarding the detection process of Human Papillomavirus (HPV) DNA probe. HPV is the most common virus that infected to human by a sexually transmitted virus. The most common high-risk HPV are 16 and 18. Interdigitated electrode (IDE) device used as based of Titanium Dioxide (TiO2) acts as inorganic surface, where by using APTES as a linker between inorganic surface and organic surface. A strategy of rapid and sensitive for the HPV detection was proposed by integrating simple DNA extraction with a gene of DNA. The extraction of the gene of DNA will make an efficiency of the detection process. It will depend on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization processes are characterized by current voltage (I-V) measurement by using KEITHLEY 6487. This strategy will perform a good sensitivity of HPV detection.

  2. A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases.

    PubMed

    Dontschuk, Nikolai; Stacey, Alastair; Tadich, Anton; Rietwyk, Kevin J; Schenk, Alex; Edmonds, Mark T; Shimoni, Olga; Pakes, Chris I; Prawer, Steven; Cervenka, Jiri

    2015-01-01

    Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene. PMID:25800494

  3. A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases

    NASA Astrophysics Data System (ADS)

    Dontschuk, Nikolai; Stacey, Alastair; Tadich, Anton; Rietwyk, Kevin J.; Schenk, Alex; Edmonds, Mark T.; Shimoni, Olga; Pakes, Chris I.; Prawer, Steven; Cervenka, Jiri

    2015-03-01

    Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene.

  4. Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes.

    PubMed

    Jetson, Rachael R; Krusemark, Casey J

    2016-08-01

    A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection-induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing. PMID:27355201

  5. Electrical switching to probe complex phases in a frustrated manganite

    NASA Astrophysics Data System (ADS)

    Asthana, Saket; Fujiwara, Kohei; Tanaka, Hidekazu

    2014-06-01

    Electrical switching was used to investigate complex phases induced by Cr-substitution in (Pr1/3Sm2/3)2/3Sr1/3MnO3. This system was expected to transform from a Type I (Mn4+/Mn3+≈3/7) to Type II (Mn4+/Mn3+≈1) manganite at critical Cr content, satisfying a virtual Mn4+/Mn3+ ratio close to unity. The phase diagram of (Pr1/3Sm2/3)2/3Sr1/3Mn0.8Cr0.2O3 including charge/spin ordered/disordered phases was probed by electrical switching. The ferromagnetic insulating phase at <~100 K, located next to the charge-ordered antiferromagnetic phase, exhibited a sudden rise in conductivity upon electric-field biasing. This resulted from the melting of charge ordering, and demonstrated the presence of a crossover regime of two coexisting magnetic orderings.

  6. Spectroscopic study one thiosemicarbazone derivative with ctDNA using ethidium bromide as a fluorescence probe.

    PubMed

    Geng, Shaoguang; Wu, Qing; Shi, Lei; Cui, Fengling

    2013-09-01

    In this study, a thiosemicarbazone derivative (E)-2-((1,4-dihydroxy-9,10-anthraquinone-2-yl)methylene)-N-(4-fluorophenyl)hydrazinecarbothioamide (DAFPT) was synthesized, and the interaction of DAFPT with calf thymus DNA (ctDNA) was explored using ethidium bromide (EB) as a fluorescence probe. The binding mode between DAFPT and ctDNA was investigated by UV absorption spectroscopy, fluorescence spectroscopy and molecular docking. The fluorescence quenching mechanism of EB-ctDNA by DAFPT might be a combined quenching type. Thermodynamic parameters showed that the reaction was spontaneous. According to ionic strength, fluorescence polarization and melting temperature (T(m)) curve results, DAFPT-ctDNA interaction was groove binding. The molecular modeling results indicated that DAFPT could slide into the A-T rich region of ctDNA. PMID:23769721

  7. Effects of DNA probe and target flexibility on the performance of a "signal-on" electrochemical DNA sensor.

    PubMed

    Wu, Yao; Lai, Rebecca Y

    2014-09-01

    We report the effect of the length and identity of a nontarget binding spacer in both the probe and target sequences on the overall performance of a folding-based electrochemical DNA sensor. Six near-identical DNA probes were used in this study; the main differences between these probes are the length (6, 10, or 14 bases) and identity (thymine (T) or adenine (A)) of the spacer connecting the two target binding domains. Despite the differences, the signaling mechanism of these sensors remains essentially the same. The methylene blue (MB)-modified probe assumes a linear unstructured conformation in the absence of the target; upon hybridization to the target, the probe adopts a "close" conformation, resulting in an increase in the MB current. Among the six sensors, the T14 and A14 sensors showed the largest signal increase upon target hybridization, highlighting the significance of probe flexibility on sensor performance. In addition to the target without a midsequence spacer, 12 other targets, each with a different oligo-T or oligo-A spacer, were used to elucidate the effect of target flexibility on the sensors' signaling capacity. For all six sensors, hybridization to targets with a 2- or 3-base spacer resulted in the largest signal increase. Higher signal enhancement was also observed with targets with an oligo-A spacer. For this sensor design, addition of a long nontarget binding spacer to the probe sequence is advantageous, as it provides flexibility for optimal target capture. The length of the spacer in the target sequence, however, should be adequately long to enable efficient hybridization yet does not introduce undesirable electrostatic and crowding effects. PMID:25110351

  8. Carrier mobility characterization of DNA-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Lin, Ting-Yu; Hung, Yu-Chueh

    2012-02-01

    Deoxyribonucleic acid (DNA) biopolymer has been emerging as a promising material for photonic applications. As many optoelectronic devices rely on carrier transportation to achieve desired functionality, carrier mobility is important for the exploitation of these biopolymer-based materials for practical implementation. In this study, we present the mobility measurement by employing time-of-flight technique and characterize the current-voltage (I-V) properties based on DNA-surfactant complexes. An additional NPB layer was introduced in the fabricated structure to serve as a charge generation layer (CGL). The dependency of hole mobility with respect to the applied electric field was characterized and a linear correlation was exhibited. Hole transport was found to be dispersive, indicating a high degree energetic disorder in these DNA-surfactant complexes. The characterization results show promises for the employment of DNA complexes in the applications of organic light-emitting devices and organic field-effect transistors.

  9. DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

    PubMed Central

    Beierlein, Frank R.; Paradas Palomo, Miguel; Sharapa, Dmitry I.; Zozulia, Oleksii; Mokhir, Andriy; Clark, Timothy

    2016-01-01

    Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries. PMID:27467071

  10. Programmed self-assembly of complex DNA nanostructures

    NASA Astrophysics Data System (ADS)

    Tian, Cheng

    DNA has served as an excellent building block to self-assemble into a wide range of one-dimensional (1D), two-dimensional (2D) and three-dimensional (3D) structures with the bottom-up method. Due to the specificity of base pairing, the DNA assembly system is predictable and robust. These DNA structures with higher diversity and complexity have potential applications as templates to organize guest molecules or nanoparticles for the nanofabrication, as biosensors for the genetic diagnosis and environmental detection, and as nanocarriers to deliver and release drugs for the therapy. My major researches focus on designing a novel building block and assembly strategies to self-assemble DNA into complex nanostructures to increase the diversity and complexity. A novel building block was first constructed, which is a parallel, left-handed DNA helix containing multiple domains of half-turn-long standard B-DNA. Such a structure can be used to introduce left-handed crossings in order to increase the diversity and complexity of DNA nanostructures, and can be taken into consideration when predicting the secondary structure of DNA/RNA molecules in cells. In addition, a tile-based directed self-assembly strategy was developed to construct DNA nanocages. In this strategy, directing building blocks were employed to control the self-assembly process of assembly building blocks. This strategy greatly expands the scope of accessible DNA nanostructures and would facilitate technological applications such as nano-guest encapsulation, drug delivery, and nanoparticle organization. As the complexity of DNA nanostructures increases, more errors might be involved in the assembly process. Therefore, a simplified design system based on T-junction was designed to build DNA arrays and minimize the assembly errors. In such system, due to the sequence symmetry, only one DNA single strand is employed and assembled into predesigned 1D and 2D arrays. This design system can be applied to assemble a

  11. Statistical mechanics of topologically constrained DNA and nucleoprotein complexes

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan Michael

    A complex connection exists between the 3 dimensional topological state of DNA in living organisms and biological processes including gene expression, DNA replication, recombination and repair. A significant limitation in developing a detailed, quantitative understanding of this connection is due to a lack of rigorous methods to calculate statistical mechanical properties of DNA molecules with complex topologies, including supercoiling, looping and knotting. This dissertation's main focus is on developing such methods and applying them to realistic DNA and nucleoprotein models. In chapter 2, a method is presented to calculate free energies and J factors of protein mediated DNA loops by normal mode analysis (NMA). This method is similar to calculations performed previously but with several significant advances. We apply the method to the specific case of DNA looping mediated by Cre recombinase protein. J factors calculated by our method are compared to experimental measurements to extract geometric and elastic properties of the Cre-DNA synaptic complex. In particular, the results suggest the existence of a synaptic complex that is more flexible than previously expected and may be explained by a stable intermediate in the reaction pathway that deviates significantly from the planar crystal structure. Calculating free energies of DNA looping is difficult in general, especially when considering intermediate length scales such as plasmid sized DNA which may readily adopt multiple topological states. In chapter 3, a novel method is presented to obtain free energies of semiflexible biopolymers with fixed topologies and arbitrary ratios of contour length L to persistence length P. High accuracy is demonstrated by calculating free energies of specific DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex

  12. Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

    PubMed Central

    2010-01-01

    Background Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes. Results First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently. Conclusions We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to

  13. Development of a DNA-Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins.

    PubMed

    Bai, Xue; Lu, Congcong; Jin, Jin; Tian, Shanshan; Guo, Zhenchang; Chen, Pu; Zhai, Guijin; Zheng, Shuzhen; He, Xiwen; Fan, Enguo; Zhang, Yukui; Zhang, Kai

    2016-07-01

    Histone post-translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA-templated technology and a photo-crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications. PMID:27169517

  14. Toehold enabling stem-loop inspired hemiduplex probe with enhanced sensitivity and sequence-specific detection of tumor DNA in serum.

    PubMed

    Yang, Fan; Wang, Siqi; Zhang, Yulin; Tang, Lina; Jin, Dan; Ning, Yong; Zhang, Guo-Jun

    2016-08-15

    The sensitivity of structure-switchable electrochemical DNA (E-DNA) sensors is generally limited by the irremovable redox labels that are close to or distant from the sensing interface. To address this issue, we design a semiduplex probe inspired by the stem-loop structure, in which the "nicked loop" domain can serve as toehold to mediate a target-responsive strand-displacement reaction. Such a reaction can fundamentally eliminate the post-responsive background current that arises from the irremovable probe, and thus improve the sensitivity. This novel toehold E-DNA (tE-DNA) sensor is able to achieve a detection limit as low as 0.2pM, which is lower than that of the classic stem-loop structured sensor by two orders of magnitude. Moreover, the toehold domain endows the sensor an excellent selectivity against a single-base mismatched sequence and high binding kinetics. By combining this heterogeneous surface-based dynamic self-assembly design with a homogeneous enzyme amplification strategy, the sensitivity can be further improved by three orders of magnitude to sub-femtomolar level. Additionally, this unique biosensor presents reliable reusability, and is capable of probing low abundance of target DNA directly in complex matrices, such as human serum, with minimal interference. These advantages make our tE-DNA sensor a promising contender in the E-DNA sensor family for clinical diagnostics. PMID:27040528

  15. DNA and buffers: the hidden danger of complex formation.

    PubMed

    Stellwagen, N C; Gelfi, C; Righetti, P G

    2000-08-01

    The free solution electrophoretic mobility of DNA differs significantly in different buffers, suggesting that DNA-buffer interactions are present in certain buffer systems. Here, capillary and gel electrophoresis data are combined to show that the Tris ions in Tris-acetate-EDTA (TAE) buffers are associated with the DNA helix to approximately the same extent as sodium ions. The borate ions in Tris-borate-EDTA (TBE) buffers interact with DNA to form highly charged DNA-borate complexes, which are stable both in free solution and in polyacrylamide gels. DNA-borate complexes are not observed in agarose gels, because of the competition of the agarose gel fibers for the borate residues. The resulting agarose-borate complexes increase the negative charge of the agarose gel fibers, leading to an increased electroendosmotic flow of the solvent in agarose-TBE gels. The combined results indicate that the buffers in which DNA is studied cannot automatically be assumed to be innocuous. PMID:10861374

  16. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  17. 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.

  18. 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

  19. DNA-based digital tension probes reveal integrin forces during early cell adhesion

    PubMed Central

    Zhang, Yun; Ge, Chenghao; Zhu, Cheng; Salaita, Khalid

    2014-01-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure due to a lack of methods for molecular force imaging. Here, to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20–30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA-hairpins with tunable force response thresholds, ligands, and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized-RGD over linear-RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial-resolution limitations of traction force microscopy. PMID:25342432

  20. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao

    2012-03-01

    This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the

  1. 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

  2. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-07-01

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the

  3. Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups.

    PubMed

    Monot, Julien; Petit, Marc; Lane, Sarah M; Guisle, Isabelle; Léger, Jean; Tellier, Charles; Talham, Daniel R; Bujoli, Bruno

    2008-05-14

    Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein. PMID:18407629

  4. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters.

    PubMed

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-08-14

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection. PMID:27406901

  5. Quantum Dot-Bead-DNA Probe-Based Hybridization Fluorescence Assays on Microfluidic Chips.

    PubMed

    Ankireddy, Seshadri Reddy; Kim, Jongsung

    2015-10-01

    The development of chip-based, quantum dot (QD)-bead-DNA conjugate probes for hybridization detection is a prime research focus in the field of microfluidics. QD-Bead-DNA probe-based hybridization detection methods are often called "bead-based assays," and their success is substantially influenced by the dispensing and manipulation capabilities of microfluidic technology. Met was identified as a prognostic marker in different cancers including lung, renal, liver, head and neck, stomach, and breast. In this report, the cancer causing Met gene was detected with QDs attached to polystyrene microbeads. We constructed a microfluidic platform using a flexible PDMS polymer. The chip consists of two channels, with two inlets and two outlets. The two channels were integrated with QD-bead-DNA probes for simultaneous detection of wild type target DNA and mutant DNA, containing three nucleotide changes compared to the wild type sequence. The fluorescence quenching ability of QDs within the channels of microfluidic chips were compared for both DNAs. PMID:26726440

  6. Binding of the anti-cancer drug daunomycin to DNA probed by second harmonic generation.

    PubMed

    Doughty, Benjamin; Rao, Yi; Kazer, Samuel W; Kwok, Sheldon J J; Turro, Nicholas J; Eisenthal, Kenneth B

    2013-12-12

    Second harmonic generation (SHG) was used to selectively probe DNA-drug interactions without the need for chemical labels or invasive detection methods. In particular, the binding constant of the anticancer drug daunomycin to a recognition triplet sequence in a 33-mer of double stranded DNA was determined. The SHG method, which is interface selective, probed the binding of daunomycin to DNA that was tethered to the surface of colloidal microparticles suspended in aqueous solution. Probing biomolecule coated colloids is expected to yield larger SH signals and provides experimental flexibility as compared to experiments performed at planar interfaces. The change in SHG intensity as daunomycin was added to the microparticle solution was fit to a Langmuir binding model, which yielded an equilibrium constant of 2.3 (±0.7) × 10(5) M(-1); the corresponding Gibbs free energy change at 20 °C is -7.2 ± 0.2 kcal/mol. Control experiments established that daunomycin preferentially binds to DNA at the recognition sequence. The equilibrium was found to be unaffected by the presence of free DNA in solution, and hyper-Rayleigh scattering from bulk molecules did not change with increasing daunomycin concentration. The extracted equilibrium constants are in agreement with the range of reported values found in the literature. PMID:23414337

  7. Effectiveness, against tuberculosis, of pseudo-ternary complexes: peptide-DNA-cationic liposome.

    PubMed

    Rosada, Rogério Silva; Silva, Célio Lopes; Santana, Maria Helena Andrade; Nakaie, Clóvis Ryuichi; de la Torre, Lucimara Gaziola

    2012-05-01

    We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery. PMID:21999959

  8. DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

    PubMed

    Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I

    2016-03-01

    Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196

  9. The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe.

    PubMed

    Shi, Chao; Shang, Fanjin; Pan, Mei; Liu, Sen; Ma, Cuiping

    2016-06-15

    Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT). PMID:26803414

  10. Demand for DNA probe testing in three genetic centres in Britain (August 1986 to July 1987).

    PubMed

    Rona, R J; Swan, A V; Beech, R; Prentice, L; Reynolds, A; Wilson, O; Mole, G; Vadera, P

    1989-04-01

    We report a preliminary analysis of the data collected during the first year of the evaluation of clinical genetics in the context of DNA probes in three genetic centres, to show the pattern of the demand for genetic services in the three centres and the services used in meeting that demand. The analysis includes information on 10,185 persons from 2852 families. The results are presented according to mode of inheritance and according to the most common disorders for which DNA probes have been used in the three centres. The results indicate that the use of DNA probes is now a major element of activity in genetic departments, and that as long as indirect DNA probe testing is the predominant manner of using recombinant technology, the clinical input will be an important element of the costs, probably more so than that of the DNA laboratories, as a large number of family members needs to be tested. In most cases centres have concentrated activity on DNA testing for common and severe genetic disorders. However, there are disorders, such as familial hypercholesterolaemia, which have not been part of the established pattern of services. Conversely, a relatively high number of families have been studied for some disorders of very low incidence. This suggests that the number of DNA laboratories should be limited. The precise arrangements will need to be established. With such services the distribution of DNA testing facilities for different disorders can be controlled to limit duplication. The model followed in Scotland based on collaboration between centres is worth considering. We have detected very large differences in take up rate of services within and between regions. Although many factors may contribute to these differences, ease of access and lay and professional awareness are probably the most important. This is supported by the fact that more patients from the same or neighbouring DHAs attend the genetic centre than from those further away. We also concluded that