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Sample records for dna complexes probed

  1. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  2. Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.

    PubMed

    Crawford, A M; Buchanan, F C; Fraser, K M; Robinson, A J; Hill, D F

    1991-01-01

    In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.

  3. Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

    NASA Astrophysics Data System (ADS)

    He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

    2011-11-01

    The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

  4. A review of scanning probe microscopy investigations of liposome-DNA complexes.

    PubMed

    Mozafari, M R; Reed, C J; Rostron, C; Hasirci, V

    2005-01-01

    Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.

  5. 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.

  6. 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

  7. 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.

  8. 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

    2015-08-07

    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

  9. Ligand-incorporation site in 5-methylcytosine-detection probe modulating the site of osmium complexation with the target DNA.

    PubMed

    Sugizaki, Kaori; Nakamura, Akiko; Yanagisawa, Hiroyuki; Okamoto, Akimitsu

    2012-09-01

    ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross-link with 5-methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine-tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5-methylcytosine-selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5-methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine-tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5-methylcytosine.

  10. 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.

  11. 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.

  12. The ODN probes conjugating the Cu(II) complex enhance the luminol chemiluminescence by assembling on the DNA template.

    PubMed

    Taniguchi, Yosuke; Nitta, Akiko; Park, Sun Min; Kohara, Akiko; Uzu, Takahiro; Sasaki, Shigeki

    2010-12-15

    Potent peroxidase-like activity of the β-ketoenamine (1)-dicopper (II) complex (2) for the chemiluminescence (CL) of luminol either in the presence or absence of H(2)O(2) has been previously demonstrated by our group. In this study, the β-ketoenamine (1) as the ligand unit for copper(II) was incorporated into the oligonucleotide (ODN) probes. It has been shown that the catalytic activity of the ODN probes conjugating the ligand-Cu(II) complex is activated by hybridization with the target DNA with the complementary sequence. Thus, this study has successfully demonstrated the basic concept for the sensitive detection of nucleic acids by CL based on the template-inductive activation of the catalytic unit for CL.

  13. DAPI: a DNA-specific fluorescent probe.

    PubMed

    Kapuscinski, J

    1995-09-01

    DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

  14. 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.

  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. Electrochemiluminescence Biosensor Based on 3-D DNA Nanomachine Signal Probe Powered by Protein-Aptamer Binding Complex for Ultrasensitive Mucin 1 Detection.

    PubMed

    Jiang, Xinya; Wang, Haijun; Wang, Huijun; Zhuo, Ying; Yuan, Ruo; Chai, Yaqin

    2017-04-04

    Herein, we fabricated a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of mucin 1 (MUC1) based on a three-dimensional (3-D) DNA nanomachine signal probe powered by protein-aptamer binding complex. The assembly of 3-D DNA nanomachine signal probe achieved the cyclic reuse of target protein based on the protein-aptamer binding complex induced catalyzed hairpin assembly (CHA), which overcame the shortcoming of protein conversion with enzyme cleavage or polymerization in the traditional examination of protein. In addition, CoFe2O4, a mimic peroxidase, was used as the nanocarrier of the 3-D DNA nanomachine signal probe to catalyze the decomposition of coreactant H2O2 to generate numerous reactive hydroxyl radical OH(•) as the efficient accelerator of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) ECL reaction to amplify the luminescence signal. Simultaneously, the assembly of 3-D DNA nanomachine signal probe was executed in solution, which led to abundant luminophore ABEI be immobilized around the CoFe2O4 surface with amplified ECL signal output since the CHA reaction was occurred unencumberedly in all directions under homogeneous environment. The prepared ECL biosensor showed a favorable linear response for MUC1 detection with a relatively low detection limit of 0.62 fg mL(-1). With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in trace protein determination.

  17. DNA interstrand cross-links of an antitumor trinuclear platinum(II) complex: thermodynamic analysis and chemical probing.

    PubMed

    Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

    2011-06-06

    The trinuclear platinum compound [{trans-PtCl(NH(3))(2)}(2)(μ-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](4+) (BBR3464) belongs to the polynuclear class of platinum-based anticancer agents. These agents form in DNA long-range (Pt,Pt) interstrand cross-links, whose role in the antitumor effects of BBR3464 predominates. Our results show for the first time that the interstrand cross-links formed by BBR3464 between two guanine bases in opposite strands separated by two base pairs (1,4-interstrand cross-links) exist as two distinct conformers, which are not interconvertible, not only if these cross-links are formed in the 5'-5', but also in the less-usual 3'-3' direction. Analysis of the conformers by differential scanning calorimetry, chemical probes of DNA conformation, and minor groove binder Hoechst 33258 demonstrate that each of the four conformers affects DNA in a distinctly different way and adopts a different conformation. The results also support the thesis that the molecule of antitumor BBR3464 when forming DNA interstrand cross-links may adopt different global structures, including different configurations of the linker chain of BBR3464 in the minor groove of DNA. Our findings suggest that the multiple DNA interstrand cross-links available to BBR3464 may all contribute substantially to its cytotoxicity.

  18. Direct Detection of Mycobacterium tuberculosis Complex DNA and Rifampin Resistance in Clinical Specimens from Tuberculosis Patients by Line Probe Assay▿

    PubMed Central

    Traore, Hamidou; van Deun, Armand; Shamputa, Isdore Chola; Rigouts, Leen; Portaels, Françoise

    2006-01-01

    The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n = 160) and previously treated (n = 260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB core region was amplified by nested PCR. The results were analyzed in conjunction with those obtained by Ziehl-Neelsen (ZN) microscopy and by culture on solid media. The LiPA test was positive for M. tuberculosis complex DNA in 389 (92.9%) specimens, including 92.0% (286 of 311) ZN-positive and 94.5% (103 of 109) ZN-negative specimens. Of these, 30.6% were RMP resistant. In contrast, 74.3% of the specimens were positive for M. tuberculosis by culture, and 30.8% of them were RMP resistant. LiPA detected M. tuberculosis complex DNA in 92.4% (110 of 119) of the culture-positive and 100.0% (41 of 41) of the culture-negative specimens from untreated patients. There was a 99.6% concordance between the RMP resistance as determined by culture and by the LiPA test. With an optimal DNA extraction method, LiPA allows rapid detection of M. tuberculosis complex DNA and RMP resistance directly from sputum specimens. LiPA can still provide useful information when culture fails for various reasons. The rapid availability of this information is necessary to adjust patient treatment and avoid the risk of amplification of drug resistance. PMID:17035487

  19. Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties.

    PubMed

    Grueso, E; López-Pérez, G; Castellano, M; Prado-Gotor, R

    2012-01-01

    The binding of [Ru(PDTA-H(2))(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.

  20. Quenching of the electrochemiluminescence of RU-complex tagged shared-stem hairpin probes by graphene oxide and its application to quantitative turn-on detection of DNA.

    PubMed

    Huang, Xiang; Huang, Xiaopeng; Zhang, An; Zhuo, Bangrong; Lu, Fushen; Chen, Yaowen; Gao, Wenhua

    2015-08-15

    Efficient and stable quenching of electrochemiluminescence (ECL) of tris(2,2'-bipyridine)-ruthenium(II) (Ru(bpy)3(2+))/tri-n-propylamine (TPrA) system by graphene oxide (GO) at the glassy carbon electrode (GCE) was reported. For figuring out the possible reasons of the quenching mechanism, the electrochemical and ECL performance of GO, different reduction degree of reduced graphene oxide (RGOs) and polymer wrapped GO modified GCEs were systematacially investigated. The results demonstrated that the oxygen-containing groups and poor electrical conductivity of GO, along with the distance between GO and Ru(bpy)3(2+) was suggested as the reasons for quenching ECL. On the basis of this essential quenching mechanism, a novel "signal on" ECL DNA biosensor for ultrasensitive detection of specific DNA sequence was constructed by self-assembling the ECL probe of thiolated shared-stem hairpin DNA (SH-DNA) tagged with Ru complex (Ru(bpy)3(2+) derivatives) on the surface of GO/gold nanoparticles (AuNPs) modified GCE. The ECL probe sequences have their ECL signal efficiently quenched when they are self-assembled on the surface of GO unless they hybridizes with their target DNA (t-DNA) sequence. The designed ECL biosensor exhibited excellent stability and reproducibility, outstanding selectivity, and an extremely sensitive response to t-DNA in a wide linear range of 100 aM-10 pM with a low detection limit of 65 aM. Our findings and the design of biosensing switch would open a new avenue in the application of GO based ECL quenching strategy for ultrasensitive bioassays.

  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. 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.

  4. 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.

  5. 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.

  6. 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

  7. Universal microbial diagnostics using random DNA probes

    PubMed Central

    Aghazadeh, Amirali; Lin, Adam Y.; Sheikh, Mona A.; Chen, Allen L.; Atkins, Lisa M.; Johnson, Coreen L.; Petrosino, Joseph F.; Drezek, Rebekah A.; Baraniuk, Richard G.

    2016-01-01

    Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD’s unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics. PMID:27704040

  8. 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.

  9. 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

  10. 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

  11. Development of a diagnostic test for Johne's disease using a DNA hybridization probe.

    PubMed Central

    Hurley, S S; Splitter, G A; Welch, R A

    1989-01-01

    A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete. Images PMID:2768445

  12. Probing the DNA Structural Requirements for Facilitated Diffusion

    PubMed Central

    2015-01-01

    DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein. PMID:25495964

  13. Scanning probe and nanopore DNA sequencing: core techniques and possibilities.

    PubMed

    Lund, John; Parviz, Babak A

    2009-01-01

    We provide an overview of the current state of research towards DNA sequencing using nanopore and scanning probe techniques. Additionally, we provide methods for the creation of two key experimental platforms for studies relating to nanopore and scanning probe DNA studies: a synthetic nanopore apparatus and an atomically flat conductive substrate with stretched DNA molecules.

  14. 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).

  15. Probing Mercury(II)-DNA Interactions by Nanopore Stochastic Sensing

    PubMed Central

    Wang, Guihua; Zhao, Qitao; Kang, Xiaofeng; Guan, Xiyun

    2013-01-01

    In this work, DNA-Hg(II) interactions were investigated by monitoring the translocation of DNA hairpins in a protein ion channel in the absence and presence of metal ions. Our experiments demonstrate that target-specific hairpin structures could be stabilized much more significantly by mercuric ions than by the stem length and the loop size of the hairpin due to the formation of Thymine-Hg(II)-Thymine complexes. In addition, the designed DNA probe allows the development of a highly sensitive nanopore sensor for Hg2+ with a detection limit of 25 nM. Further, the sensor is specific, and other tested metal ions including Pb2+, Cu2+, Cd2+, etc. with concentrations of up to two orders of magnitude greater than that of Hg2+ would not interfere with the mercury detection. PMID:23565989

  16. 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.

  17. 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.

  18. Electroactive crown ester-Cu(2+) complex with in-situ modification at molecular beacon probe serving as a facile electrochemical DNA biosensor for the detection of CaMV 35s.

    PubMed

    Zhan, Fengping; Liao, Xiaolei; Gao, Feng; Qiu, Weiwei; Wang, Qingxiang

    2017-06-15

    A novel electrochemical DNA biosensor has been facilely constructed by in-situ assembly of electroactive 4'-aminobenzo-18-crown-6-copper(II) complex (AbC-Cu(2+)) on the free terminal of the hairpin-structured molecule beacon. The 3'-SH modified molecule beacon probe was first immobilized on the gold electrode (AuE) surface through self-assembly chemistry of Au-S bond. Then the crow ester of AbC was covalently coupled with 5'-COOH on the molecule beacon, and served as a platform to attach the Cu(2+) by coordination with ether bond (-O-) of the crown cycle. Thus, an electroactive molecule beacon-based biosensing interface was constructed. In comparison with conventional methods for preparation of electroactive molecule beacon, the approach presented in this work is much simpler, reagent- and labor-saving. Selectivity study shows that the in-situ fabricated electroactive molecule beacon remains excellent recognition ability of pristine molecule beacon probe to well differentiate various DNA fragments. The target DNA can be quantatively determined over the range from 0.10pM to 0.50nM. The detection limit of 0.060pM was estimated based on signal-to-noise ratio of 3. When the biosensor was applied for the detection cauliflower mosaic virus 35s (CaMV 35s) in soybean extraction samples, satisfactory results are achieved. This work opens a new strategy for facilely fabricating electrochemical sensing interface, which also shows great potential in aptasensor and immurosensor fabrication.

  19. 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.

  20. 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.

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

    PubMed

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

    2010-03-29

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

  2. 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.

  3. Advanced Molecular Probes for Sequence-Specific DNA Recognition

    NASA Astrophysics Data System (ADS)

    Bertucci, Alessandro; Manicardi, Alex; Corradini, Roberto

    DNA detection can be achieved using the Watson-Crick base pairing with oligonucleotides or oligonucleotide analogs, followed by generation of a physical or chemical signal coupled with a transducer device. The nature of the probe is an essential feature which determines the performances of the sensing device. Many synthetic processes are presently available for "molecular engineering" of DNA probes, enabling label-free and PCR-free detection to be performed. Furthermore, many DNA analogs with improved performances are available and are under development; locked nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs, morpholino oligonucleotides (MO) and other modified probes have shown improved properties of affinity and selectivity in target recognition compared to those of simple DNA probes. The performances of these probes in sensing devices, and the requirements for detection of unamplified DNA will be discussed in this chapter. Chemistry and architectures for conjugation of probes to reporter units, surfaces and nanostructures will also be discussed. Examples of probes used in ultrasensitive detection of unamplified DNA are listed.

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

    PubMed

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

    2015-06-01

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

  6. 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

  7. DNA nanostructure-based imaging probes and drug carriers.

    PubMed

    Zhan, Pengfei; Jiang, Qiao; Wang, Zhen-Gang; Li, Na; Yu, Haiyin; Ding, Baoquan

    2014-09-01

    Self-assembled DNA nanostructures are well-defined nanoscale shapes, with uniform sizes, precise spatial addressability, and excellent biocompatibility. With these features, DNA nanostructures show great potential for biomedical applications; various DNA-based biomedical imaging probes or payload delivery carriers have been developed. In this review, we summarize the recent developments of DNA-based nanostructures as tools for diagnosis and cancer therapy. The biological effects that are brought about by DNA nanostructures are highlighted by in vitro and in vivo imaging, antitumor drug delivery, and immunostimulatory therapy. The challenges and perspectives of DNA nanostructures in the field of nanomedicine are discussed.

  8. 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

  9. 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.

  10. Fluorescent cyanine probe for DNA detection and cellular imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Yong-Chao; Zheng, Mei-Ling; Zhao, Zhen-Sheng; Duan, Xuan-Ming

    2014-03-01

    In our study, two carbazole-based cyanines, 3,6-bis[2-(1-methylpyridinium)vinyl]-9-methyl carbazole diiodide (A) and 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3yl)methane diiodide (B) were synthesized and employed as light-up probes for DNA and cell imaging. Both of the cyanine probes possess a symmetric structure and bis-cationic center. The obvious induced circular dichroism signals in circular dichroism spectra reveal that the molecules can specifically interact with DNA. Strong fluorescence enhancement is observed when these two cyanines are bound to DNA. These cyanine probes show high binding affinity to oligonucleotides but different binding preferences to various secondary structures. Confocal microscopy images of fixed cell stained by the probes exhibit strong brightness and high contrast in nucleus with a very low cytoplasmic background.

  11. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    PubMed

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  12. 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.

  13. [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.

  14. Probing the elastic limit of DNA bending.

    PubMed

    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.

  15. 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.

  16. 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

  17. DNA hybridization probe for clinical diagnosis of Entamoeba histolytica.

    PubMed Central

    Samuelson, J; Acuna-Soto, R; Reed, S; Biagi, F; Wirth, D

    1989-01-01

    As an alternative to microscopic identification of Entamoeba histolytica parasites isolated from stool, a sensitive and species-specific DNA hybridization probe was made for rapid diagnosis of E. histolytica parasites in clinical samples directly applied to nylon membranes. The DNA hybridization probe was made by screening a genomic library of a virulent HM-1:IMSS strain of E. histolytica to detect recombinant plasmids containing highly repeated parasite DNA sequences. Four plasmid clones that reacted across Entamoeba species coded for highly repeated rRNA genes of E. histolytica. Four other plasmid clones were E. histolytica specific in that they bound to four axenized and nine xenic strains of E. histolytica but did not recognize closely related E. histolytica-like Laredo, Entamoeba moshkovskii, or Entamoeba invadens parasites. The diagnostic clones detected as few as eight cultured amoebae and did not distinguish between pathogenic and nonpathogenic zymodemes of E. histolytica. The diagnostic clones were sequenced and contained 145-base-pair sequences which appear to be tandemly repeated in the genome. No stable transcript which is homologous to the diagnostic DNA was detected. In a study of stool samples from Mexico City shown by microscopy to contain E. histolytica, Entamoeba coli, Giardia lamblia, Endolimax nana, Trichuris trichiuria, and Chilomastix mesnili parasites, the DNA hybridization probe demonstrated a sensitivity of 1.0 and a specificity of 0.93. We conclude that the DNA hybridization probe can be used for rapid and accurate diagnosis of E. histolytica parasites. Images PMID:2542361

  18. Mixed DNA/Oligo(ethylene glycol) Functionalized Gold Surface Improve DNA Hybridization in Complex Media

    SciTech Connect

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

    2006-01-01

    Reliable, direct 'sample-to-answer' capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s{yields}{pi}* transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein

  19. 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.

  20. DNA fingerprints of farm animals generated by microsatellite and minisatellite DNA probes.

    PubMed

    Haberfeld, A; Cahaner, A; Yoffe, O; Plotsky, Y; Hillel, J

    1991-01-01

    A multi-locus DNA probe, R18.1, derived from a bovine genomic library, detected DNA fingerprints of highly polymorphic loci in hybridization to genomic DNA from poultry and sheep, and of moderate polymorphic loci in cattle and human DNA. The average numbers of detected bands in chickens and sheep were 27.8 and 21.4, and the average band sharing levels were 0.25 and 0.33, respectively. In hybridization to cattle and human DNA, the results were less polymorphic; nevertheless, individual identification is feasible using probe R18.1. The results obtained by R18.1 were compared to results obtained by Jeffreys minisatellite probe 33.6 and two microsatellite oligonucleotides, (GT)12 and (GTG)5. The total number of detected loci using probes R18.1 and 33.6 were estimated in chickens through family analysis of broilers and the maximal number of detectable loci was calculated.

  1. DNA-Templated Aptamer Probe for Identification of Target Proteins.

    PubMed

    Bi, Wenjing; Bai, Xue; Gao, Fan; Lu, Congcong; Wang, Ye; Zhai, Guijin; Tian, Shanshan; Fan, Enguo; Zhang, Yukui; Zhang, Kai

    2017-04-04

    Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

  2. Array-based electrical detection of DNA with nanoparticle probes.

    PubMed

    Park, So-Jung; Taton, T Andrew; Mirkin, Chad A

    2002-02-22

    A DNA array detection method is reported in which the binding of oligonucleotides functionalized with gold nanoparticles leads to conductivity changes associated with target-probe binding events. The binding events localize gold nanoparticles in an electrode gap; silver deposition facilitated by these nanoparticles bridges the gap and leads to readily measurable conductivity changes. An unusual salt concentration-dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. Using this method, we have detected target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of approximately 100,000:1.

  3. Evolving DNA motifs to predict GeneChip probe performance

    PubMed Central

    Langdon, WB; Harrison, AP

    2009-01-01

    Background Affymetrix High Density Oligonuclotide Arrays (HDONA) simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe. Results Regular expressions can be automatically created from a Backus-Naur form (BNF) context-free grammar using strongly typed genetic programming. Conclusion The automatically produced motif is better at predicting poor DNA sequences than an existing human generated RE, suggesting runs of Cytosine and Guanine and mixtures should all be avoided. PMID:19298675

  4. Isolation and characterization of DNA probes for human chromosome 21.

    PubMed

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  5. 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.

  6. Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes.

    PubMed

    Costa, Pedro; Amaro, Ana; Ferreira, Ana S; Machado, Diana; Albuquerque, Teresa; Couto, Isabel; Botelho, Ana; Viveiros, Miguel; Inácio, João

    2014-12-01

    Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows the assessment of the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows us to infer the species of the isolate as M. tuberculosis (if all RDs are present), Mycobacterium caprae (if only RD1 and RD4 are present) and Mycobacterium bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CI(P95%) 0.929-1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostic framework of veterinary diagnostic laboratories, with a particular focus for reference laboratories.

  7. Synthesis of a new Ni-phenanthroline complex and its application as an electrochemical probe for detection of nucleic acid.

    PubMed

    Qiu, Bin; Guo, Longhua; Guo, Chunhua; Guo, Zhiyong; Lin, Zhenyu; Chen, Guonan

    2011-01-15

    A new DNA sensor using a nickel(II) phenanthroline complex ([Ni(phen)(2)PHPIP]·2ClO(4)) as the electrochemical probe was developed. The sensor is very sensitive and selective for calf thymus DNA (ctDNA) detection in aqueous medium. The Ni-phenanthroline probe was synthesized by a two-step preparation using p-hydroxy-phenylimidazo-1,10-phenanthroline (PHPIP) as the ligand and characterized with IR, UV and MS. Some interesting electrochemical properties of the Ni-complex and the interactions of the complex with ctDNA were reported. The calculated dynamics parameters of the electrode process indicate that there are obvious interactions between the probe and the ctDNA in aqueous solution. Under constant potential conditions, the redox current peak of the probe (Ni-complex) decreases obviously as the probe interacts/binds with ctDNAs. This unexpected electrochemical behavior may suggest that a new adduct through the binding of Ni-phenanthroline complex with ctDNA is formed electrochemically. By estimation, the binding ratio of the probe and ctDNA was found to be 1:1 with a binding constant β=4.29×10(5) mol L(-1) in aqueous solution at room temperature.

  8. Probe Microscopic Studies of DNA Molecules on Carbon Nanotubes

    PubMed Central

    Umemura, Kazuo; Izumi, Katsuki; Oura, Shusuke

    2016-01-01

    Hybrids of DNA and carbon nanotubes (CNTs) are promising nanobioconjugates for nanobiosensors, carriers for drug delivery, and other biological applications. In this review, nanoscopic characterization of DNA-CNT hybrids, in particular, characterization by scanning probe microscopy (SPM), is summarized. In many studies, topographical imaging by atomic force microscopy has been performed. However, some researchers have demonstrated advanced SPM operations in order to maximize its unique and valuable functions. Such sophisticated approaches are attractive and will have a significant impact on future studies of DNA-CNT hybrids.

  9. 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.

  10. Luminescence sensitization of Tb(3+)-DNA complexes by Ag().

    PubMed

    Xu, Lijun; Zhou, Lu; Chen, Xing; Shen, Xiaoqiang; Wang, Jine; Zhang, Jianye; Pei, Renjun

    2017-03-03

    Terbium ions (Tb(3+)) with unique photophysical properties have been utilized to develop biosensors with low background and high sensitivity. In this study, the Ag(+)-sensitized luminescence of Tb(3+)-DNA complexes was uncovered. The luminescence of Tb(3+)-DNA complexes could be enhanced by more than 30 times in the presence of Ag(+), when Tb(3+) was bound with poly(G) and poly(T) whereas not with other homopolymers. This research confirmed that the sensitization resulted from the interaction of Ag(+) with certain bases involved in DNA, not just with the reported certain G-quadruplex sequence. The coordination of Ag(+) to guanine and thymine bases was expected to increase their rigidities, form Tb(3+)-DNA-Ag(+) ternary structures, and thus enhance energy transfer from guanine and thymine to Tb(3+). These findings benefit the development of sensitive luminescence probes for various nucleic acids-related targets.

  11. Probing Nucleosome Stability with a DNA Origami Nanocaliper.

    PubMed

    Le, Jenny V; Luo, Yi; Darcy, Michael A; Lucas, Christopher R; Goodwin, Michelle F; Poirier, Michael G; Castro, Carlos E

    2016-07-26

    The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

  12. Probe mapping to facilitate transposon-based DNA sequencing

    SciTech Connect

    Strausbaugh, L.D.; Bourke, M.T.; Sommer, M.T.; Coon, M.E.; Berg, C.M. )

    1990-08-01

    A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. The authors demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon {gamma}{delta} (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal {gamma}{delta} segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine {gamma}{delta} insertion sites. The insertions were quire randomly distributed, even though the target DNA fragment had both A+T-rich and G+C-rich regions; in G+C-rich DNA, the insertions were found in A+T-rich valleys. These data demonstrate that {gamma}{delta} is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

  13. Identification and epidemiological typing of Naegleria fowleri with DNA probes.

    PubMed Central

    Kilvington, S; Beeching, J

    1995-01-01

    Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment. PMID:7793928

  14. DNA probe and PCR-specific reaction for Lactobacillus plantarum.

    PubMed

    Quere, F; Deschamps, A; Urdaci, M C

    1997-06-01

    A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.

  15. Scanning Probe Microscopy of DNA with a Quartz Tuning Fork

    NASA Astrophysics Data System (ADS)

    King, G. M.; Nunes, G., Jr.

    2001-03-01

    Quartz tuning-forks have recently been put to use as highly sensitive force detectors in atomic force microscopy (AFM).(F.J.Giessibl et al.), Science 289, 422 (2000). In this study we have applied a home-built, tuning-fork based AFM to the investigation of single and double stranded DNA (ssDNA and dsDNA). We operate the microscope in the non-contact mode (typical tip amplitude ~1 nm) with a variety of tips (e.g. Si, Si_3N_4, W). Here we report on recent results showing that the apparent height of plasmid dsDNA on mica substrates depends on both the tip material and imaging frequency shift. This talk will also review our efforts to probe ssDNA with a chemically functionalized tip. Current and future prospects for this dynamic-mode, chemically-sensitive force microscopy technique will be discussed.

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

    PubMed

    Maxwell, E S; Sarge, K D

    1988-11-30

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

  17. Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA

    PubMed Central

    Vinogradova, O.A.

    2010-01-01

    The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA–dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA–dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA–dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme–substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA–substrates. We also discuss relevant approaches to increasing the selectivity of enzyme–dependent reactions. These approaches involve the use of modified oligonucleotide probes which “disturb” the native structure of the DNA–substrate complexes. PMID:22649627

  18. 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.

  19. 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.

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

    PubMed Central

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

    2008-01-01

    Background 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 100 kb, careful probe selection and characterization are of paramount importance. Results 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 ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus. Conclusion 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. PMID:19108707

  1. 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.

  2. Multipyrene Tandem Probes for Point Mutations Detection in DNA

    PubMed Central

    Kholodar, Svetlana A.; Novopashina, Darya S.; Meschaninova, Mariya I.; Venyaminova, Alya G.

    2013-01-01

    Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. PMID:24455205

  3. 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.

  4. 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.

  5. Ultrafast transient absorption studies of ruthenium and rhenium dipyridophenazine complexes bound to DNA and polynucleotides

    NASA Astrophysics Data System (ADS)

    Creely, Caitriona M.; Kelly, John M.; Feeney, M. M.; Hudson, S.; Penedo, J. C.; Blau, Werner J.; Elias, B.; Kirsch-De Mesmaeker, Andree; Matousek, P.; Towrie, M.; Parker, A. W.; Dyer, J. S.; George, Mikhael W.; Coates, C. G.; McGarvey, John J.

    2003-03-01

    We report on ultrafast pump and probe studies of biological systems, in the form of polynucleotide and calf thymus DNA complexes. Molecules for study are bound to the polynucleotides and probed in the visible region to observe changes in the absorption over time. Various dipyridophenazine metal complexes are studied alone and complexed with DNA or synthetic polynucleotides to investigate changes occurring in their excited states upon interacting with nucleobases. Transient absorption measurements are performed pumping at 400nm and probing from 450-700nm with pulse duration of 400fs.

  6. DNA Probes As Potential Tools for the Detection of Marteilia refringens.

    PubMed

    Le Roux F; Audemard; Barnaud; Berthe

    1999-11-01

    Since its first description, the paramyxean parasite Marteilia refringens has been recognized as a significant pathogen of bivalve mollusks. The existence of a complex life cycle was postulated by many authors. Here we report the development of DNA-based detection assays as powerful tools to elucidate the Marteilia refringens life cycle. After alignment of the Marteilia refringens ribosomal DNA small subunit sequence with those of various eukaryotic organisms, polymerase chain reaction primers were designed. Specific primers were used to amplify DNA extracted from purified Marteilia refringens and infected hosts. The specificity of amplified fragments was confirmed by Southern blotting with an oligoprobe. For in situ hybridization, four probes were tested for specific detection of 18S rRNA isolated from Marteilia refringens and other eukaryotic cells by Northern blotting. The most specific probe, Smart 2, was successfully used to detect Marteilia refringens by in situ hybridization in infected oysters and mussels.

  7. 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.

  8. 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.

  9. 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

  10. [Diagnosed tuberculosis using specific DNA probe hybridization methods].

    PubMed

    Furuta, Itaru; Yamazumi, Toshiaki

    2002-11-01

    In Japan, reported cases of tuberculosis had declined nearly every year until 1995. However, in 1997 newly recorded cases began increasing for the first time in more than 38 years. Recent studies using DNA fingerprinting show that person- to person transmission may account for as many as one-third of new cases of tuberculosis in citizen populations. Nucleic acid hybridization methods using specific DNA probes can specifically identify M. tuberculosis and other mycobacterial species. Rapid nucleic acid amplification techniques such as polymerase chain reaction methods allow direct identification of M. tuberculosis in clinical specimens. Is 6110 has been exploited extensively as a clonal marker in molecular epidemiology studies of tuberculosis. The emergence of resistance to antituberculosis drugs is a relevant matter worldwide. A recent genotypic method allows earlier detection of RFP-resistant and INH-resistant stains using probes for mutation in rpoB and in katG.

  11. Sensitive determination of DNA based on the interaction between prulifloxacin-terbium(III) complex and DNA.

    PubMed

    Wu, Ting; Fang, Biyun; Chang, Lin; Liu, Min; Chen, Fang

    2013-01-01

    A simple spectrofluorimetric method is described for the determination of DNA, based on its enhancement of the fluorescence intensity of prulifloxacin (PUFX)-Tb(3+). The luminescence intensity of the PUFX-Tb(3+) complex increased up to 10-fold after adding DNA. The excitation and emission wavelengths were 345 and 545 nm, respectively. Under optimum conditions, variations in the fluorescence intensity showed a good linear relationship with the concentration of hsDNA in the range of 3.0 × 10(-9) to 1.0 × 10(-6) g/mL, with a correlation coefficient (R) of 0.997, and the detection limit was 2.1 × 10(-9) g/mL. The method was successfully applied to the determination of DNA in synthetic samples, and recoveries were in the range 97.3-102.0%. The mechanism of fluorescence enhancement of the PUFX-Tb(3+) complex by DNA is also discussed. The mechanism may involve formation of a ternary complex mainly by intercalation binding together with weak electrostatic interaction, which will increase the energy transition from ligand to Tb(3+), increasing the rigidity of the complex, and decreasing the radiationless energy loss through O-H vibration of the H2O molecule in the PUFX-Tb(3+) complex. Compared with the previous DNA probes, the proposed method is not only more robust and friendly to the environment, but also of relatively higher sensitivity.

  12. 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.

  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. Multivalent Lipid--DNA Complexes: Distinct DNA Compaction Regimes

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Safinya, C. R.

    2004-03-01

    Cationic liposomes (CL), while intrinsically advantageous in comparison to viruses, still have limited success for gene therapy and require more study. CL spontaneously self-assemble with DNA via counterion release, forming small particles approximately 200nm in diameter. X-ray diffraction reveals CL-DNA structures that are typically a multilamellar organization of lipids with DNA intercalated between the layers. We explore the structural properties of CL-DNA complexes formed with new multivalent lipids (Ewert et al, J. Med. Chem. 2002; 45:5023) that range from 2+ to 16+. Contrary to a simple prediction for the DNA interaxial spacing d_DNA based on a geometrical space-filling model, these lipids show dramatic DNA compaction, down to d_DNA ˜ 25 ÅVariations in the membrane charge density, σ _M, lead to distinct spacing regimes. We propose that this DNA condensation is controlled by a unique locking mechanism between the DNA double helix and the large, multivalent lipid head groups. Funded by NSF DMR-0203755 and NIH GM-59288.

  15. Large branched self-assembled DNA complexes

    NASA Astrophysics Data System (ADS)

    Tosch, Paul; Wälti, Christoph; Middelberg, Anton P. J.; Davies, A. Giles

    2007-04-01

    Many biological molecules have been demonstrated to self-assemble into complex structures and networks by using their very efficient and selective molecular recognition processes. The use of biological molecules as scaffolds for the construction of functional devices by self-assembling nanoscale complexes onto the scaffolds has recently attracted significant attention and many different applications in this field have emerged. In particular DNA, owing to its inherent sophisticated self-organization and molecular recognition properties, has served widely as a scaffold for various nanotechnological self-assembly applications, with metallic and semiconducting nanoparticles, proteins, macromolecular complexes, inter alia, being assembled onto designed DNA scaffolds. Such scaffolds may typically contain multiple branch-points and comprise a number of DNA molecules selfassembled into the desired configuration. Previously, several studies have used synthetic methods to produce the constituent DNA of the scaffolds, but this typically constrains the size of the complexes. For applications that require larger self-assembling DNA complexes, several tens of nanometers or more, other techniques need to be employed. In this article, we discuss a generic technique to generate large branched DNA macromolecular complexes.

  16. 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

  17. 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.

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

    PubMed

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

    2016-02-02

    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.

  19. 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

    2016-04-04

    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.

  20. Probing the binding mode of psoralen to calf thymus DNA.

    PubMed

    Zhou, Xiaoyue; Zhang, Guowen; Wang, Langhong

    2014-06-01

    The binding properties between psoralen (PSO) and calf thymus DNA (ctDNA) were predicted by molecular docking, and then determined with the use of UV-vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, coupled with DNA melting and viscosity measurements. The data matrix obtained from UV-vis spectra was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The pure spectra and the equilibrium concentration profiles for PSO, ctDNA and PSO-ctDNA complex extracted from the highly overlapping composite response were obtained simultaneously to evaluate the PSO-ctDNA interaction. The intercalation mode of PSO binding to ctDNA was supported by the results from the melting studies, viscosity measurements, iodide quenching and fluorescence polarization experiments, competitive binding investigations and CD analysis. The molecular docking prediction showed that the specific binding most likely occurred between PSO and adenine bases of ctDNA. FT-IR spectra studies further confirmed that PSO preferentially bound to adenine bases, and this binding decreased right-handed helicity of ctDNA and enhanced the degree of base stacking with the preservation of native B-conformation. The calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played a major role in the binding process.

  1. Using surface-enhanced Raman spectroscopy to probe for genetic markers on single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Moody, Benjamin; Leotaud, John; McCarty, Gregory S.

    2010-03-01

    Methods capable of quickly and inexpensively collecting genetic information are of increasing importance. We report a method of using surface-enhanced Raman spectroscopy to probe single-stranded DNA for genetic markers. This unique approach is used to analyze unmodified genes of moderate length for genetic markers by hybridizing native test oligonucleotides into a surface-enhanced Raman complex, vastly increasing detection sensitivity as compared to traditional Raman spectroscopy. The Raman complex is formed by sandwiching the test DNA between 40-nm gold nanoparticles and a photolithographically defined gold surface. With this design, we are able to collect characteristic Raman spectra about the test DNA and to detect genetic markers such as single-nucleotide polymorphisms (SNPs) and polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant trends regarding Raman intensity.

  2. Isolation of human minisatellite loci detected by synthetic tandem repeat probes: direct comparison with cloned DNA fingerprinting probes.

    PubMed

    Armour, J A; Vergnaud, G; Crosier, M; Jeffreys, A J

    1992-08-01

    As a direct comparison with cloned 'DNA fingerprinting' probes, we present the results of screening an ordered array Charomid library for hypervariable human loci using synthetic tandem repeat (STR) probes. By recording the coordinates of positive hybridization signals, the subset of clones within the library detected by each STR probe can be defined, and directly compared with the set of clones detected by naturally occurring (cloned) DNA fingerprinting probes. The STR probes vary in the efficiency of detection of polymorphic minisatellite loci; among the more efficient probes, there is a strong overlap with the sets of clones detected by the DNA fingerprinting probes. Four new polymorphic loci were detected by one or more of the STR probes but not by any of the naturally occurring repeats. Sequence comparisons with the probe(s) used to detect the locus suggest that a relatively poor match, for example 10 out of 14 bases in a limited region of each repeat, is sufficient for the positive detection of tandem repeats in a clone in this type of library screening by hybridization. These results not only provide a detailed evaluation of the usefulness of STR probes in the isolation of highly variable loci, but also suggest strategies for the use of these multi-locus probes in screening libraries for clones from hypervariable loci.

  3. 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.

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

    NASA Astrophysics Data System (ADS)

    Wang, Juexiao Sherry; Zhang, David Yu

    2015-07-01

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

  5. DNA Interactions with Ruthenium(ll) Polypyridine Complexes Containing Asymmetric Ligands

    PubMed Central

    Chao, Hui

    2005-01-01

    In an attempt to probe nucleic acid structures, numerous Ru(II) complexes with different ligands have been synthesized and investigated. In this contribution we focus on the DNA-binding properties of ruthenium(II) complexes containing asymmetric ligands that have attracted little attention in the past decades. The influences of the shape and size of the ligand on the binding modes, affinity, enantioselectivities and photocleavage of the complexes to DNA are described. PMID:18365086

  6. Cleavage enhancement of specific chemical bonds in DNA-Cisplatin complexes induced by X-rays

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Yao, Xiaobin; Luo, Xinglan; Fu, Xianzhi

    2014-04-01

    The chemical bond transformation of cisplatin-DNA complexes can be probed efficiently by XPS which provides a concomitant X-ray irradiation source as well. The presence to Pt could considerably increase formation of the SE induced by X-ray and that the further interaction of these LEE with DNA leads to the enhancement of bond cleavages.

  7. Antitumor effects of a tetradentate amido-carboxylate ligands and corresponding square-planar palladium(II) complexes toward some cancer cells. Crystal structure, DFT modeling and ligand to DNA probe docking simulation.

    PubMed

    Matović, Zoran D; Mrkalić, Emina; Bogdanović, Gordana; Kojić, Vesna; Meetsma, Auke; Jelić, Ratomir

    2013-04-01

    Novel square-planar palladium(II) complexes with O-N-N-O-type ligands H4mda (H4mda=malamido-N,N'-diacetic acid) and H4obp (H4obp=oxamido-N,N'-di-3-propionic acid) were prepared and characterized. The ligands coordinate to the palladium(II) ion via two pairs of deprotonated ligating atoms with square chelation. A four coordinate, square-planar geometry was verified crystallographicaly for the K2[Pd(mda)]·H2O complex. The binary and ternary systems of Pd(II) ion with H4mda or H4obp (L) as primary ligands and guanosine (A) as secondary ligand were studied in aqueous solutions in 0.1 M NaCl ionic medium at 25 °C by potentiometric titrations. In addition, calculations based on density functional methods (DFT) were carried out. A natural bonding orbital analysis indicated that the Pd-N bonds are three-centric in nature and mainly governed by charge transfer via a strong delocalization of the oxygen lone pair with "p" character into the bonding Pd-N orbital. Mononuclear palladium(II) complexes together with amido acid N,O-containing ligands were tested against several tumor cells and reveal significant antitumor activity and lower resistance of tumor cells in vitro than cisplatin. In this paper, interactions of palladium complexes with DNA are discussed in order to provide guidance and determine structure and antitumor activity relationships for continuing studies of these systems. Docking simulation on DNA dodecamer or 29-mer (Lippard solved crystal structures), suggests several favorable interactions with the hydrogen pocket/binding site for the incoming ligands. These results support amidoacids/Pd complexes as novel antitumor drugs and suggest that their potent cell life inhibition may contribute to its anti-cancer efficacy.

  8. A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

    PubMed

    Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

    2015-01-01

    A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  10. Creating complex molecular topologies by configuring DNA four-way junctions

    NASA Astrophysics Data System (ADS)

    Liu, Di; Chen, Gang; Akhter, Usman; Cronin, Timothy M.; Weizmann, Yossi

    2016-10-01

    The realization of complex topologies at the molecular level represents a grand challenge in chemistry. This necessitates the manipulation of molecular interactions with high precision. Here we show that single-stranded DNA (ssDNA) knots and links can be created by utilizing the inherent topological properties that pertain to the DNA four-way junction, at which the two helical strands form a node and can be configured conveniently and connected for complex topological construction. Using this strategy, we produced series of ssDNA topoisomers with the same sequences. By finely designing the curvature and torsion, double-stranded DNA knots were accessed by hybridizing and ligating the complementary strands with the knotted ssDNA templates. Furthermore, we demonstrate the use of a constructed ssDNA knot both to probe the topological conversion catalysed by DNA topoisomerase and to study the DNA replication under topological constraint.

  11. 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.

  12. 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

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

    PubMed

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

    2016-04-01

    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.

  14. Intercalation processes of copper complexes in DNA.

    PubMed

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

    2015-06-23

    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.

  15. 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

  16. Design space for complex DNA structures.

    PubMed

    Wei, Bryan; Dai, Mingjie; Myhrvold, Cameron; Ke, Yonggang; Jungmann, Ralf; Yin, Peng

    2013-12-04

    Nucleic acids have emerged as effective materials for assembling complex nanoscale structures. To tailor the structures to function optimally for particular applications, a broad structural design space is desired. Despite the many discrete and extended structures demonstrated in the past few decades, the design space remains to be fully explored. In particular, the complex finite-sized structures produced to date have been typically based on a small number of structural motifs. Here, we perform a comprehensive study of the design space for complex DNA structures, using more than 30 distinct motifs derived from single-stranded tiles. These motifs self-assemble to form structures with diverse strand weaving patterns and specific geometric properties, such as curvature and twist. We performed a systematic study to control and characterize the curvature of the structures, and constructed a flat structure with a corrugated strand pattern. The work here reveals the broadness of the design space for complex DNA nanostructures.

  17. Resonance light scattering method for the determination of DNA with cationic methacrylate based polymer nanoparticle probes.

    PubMed

    Zou, Qi-Chao; Zhang, Jin-Zhi; Chai, Shi-Gan

    2011-11-01

    Narrowly distributed cationic poly (methyl methacrylate-co-diacetone acrylamide) (P(MMA-DAAM)) nanoparticles were successfully prepared by microemulsion polymerization. Photon correlation spectrometer (PCS) measurement and transmission electron microscope (TEM) observation revealed that z-average particle size of P(MMA-DAAM) is ∼27.5 nm. It was found that these cationic nanoparticles interact with DNA through electrostatic interaction to form P(MMA-DAAM)-DNA complex, which significantly enhances the resonance light scattering (RLS) signal. Therefore, a novel method using this polymer nanoparticle as a new probe for the detection of DNA by RLS technique is developed in this paper. The results showed this method is very convenient, sensitive, and reproducible.

  18. Photoresponsive Supramolecular Complexes as Efficient DNA Regulator

    PubMed Central

    Cheng, Hong-Bo; Zhang, Ying-Ming; Xu, Chao; Liu, Yu

    2014-01-01

    Two supramolecular complexes of trans-1⊂CB[8] and trans-2⊂CB[8] were successfully achieved by the controlled selective complexation process of cucurbit[8]uril (CB[8]) with hetero-guest pair containing azobenzene and bispyridinium moieties in aqueous solution, exhibiting the reversibly light-driven movements of CB[8] upon the photocontrollable isomerization of azophenyl axle components. Significantly, the obtained bistable supramolecular complexes and their corresponding [2]pseudorotaxanes could act as a promising concentrator and cleavage agent to regulate the binding behaviors with DNA molecules. PMID:24572680

  19. Islands of Complex DNA Are Widespread in Drosophila Centric Heterochromatin

    PubMed Central

    Le, M. H.; Duricka, D.; Karpen, G. H.

    1995-01-01

    Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three ``islands'' of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed. PMID:8536977

  20. 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.

  1. 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.

  2. Probing the microenvironments in the grooves of Z-DNA using dan-modified oligonucleotides.

    PubMed

    Kimura, Takumi; Kawai, Kiyohiko; Majima, Tetsuro

    2006-04-14

    The environment-sensitive fluorophore dan (6-dimethylamino-2-acyl-naphthalene)- modified dC or dG bases were introduced into the Z-DNA forming sequence. It was demonstrated that both grooves of Z-DNA are more hydrated than those of B-DNA. Dan will be useful for probing the microenvironments in the grooves among the DNA polymorphs.

  3. 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.

  4. Probing the Dynamic Interaction between Damaged DNA and a Cellular Responsive Protein Using a Piezoelectric Mass Biosensor.

    PubMed

    Jin, Yulong; Xie, Yunfeng; Wu, Kui; Huang, Yanyan; Wang, Fuyi; Zhao, Rui

    2017-03-15

    The binding events between damaged DNA and recognition biomolecules are of great interest for understanding the activity of DNA-damaging drugs and the related DNA repair networks. Herein, a simple and sensitive sensor system was tailored for real-time probing of the dynamic molecular recognition between cisplatin-damaged-DNA (cisPt-DNA) and a cellular responsive protein, high-mobility-group box 1 (HMGB1). By integration of flow injection analysis (FIA) with quartz crystal microbalance (QCM), the interaction time-course of cisPt-DNA and HMGB1 domain A (HMGB1a) was investigated. The highly specific sensing interface was carefully designed and fabricated using cisPt-DNA as recognition element. A hybrid self-assembled monolayer consisting of cysteamine and mercaptohexanol was introduced to resist nonspecific adsorption. The calculated kinetic parameters (kass and kdiss) and the dissociation constant (KD) demonstrated the rapid recognition and tight binding of HMGB1a toward cisPt-DNA. Molecular docking was employed to simulate the complex formed by cisPt-DNA and HMGB1a. The tight binding of such a DNA-damage responsive complex is appealing for the downstream molecular recognition event related to the resistance to DNA repair. This continuous-flow QCM biosensor is an ideal tool for studying specific interactions between drug-damaged-DNAs and their recognition proteins in a physiological-relevant environment, and will provide a potential sensor platform for rapid screening and evaluating metal anticancer drugs.

  5. Dynamics of DNA/intercalator complexes

    NASA Astrophysics Data System (ADS)

    Schurr, J. M.; Wu, Pengguang; Fujimoto, Bryant S.

    1990-05-01

    Complexes of linear and supercoiled DNAs with different intercalating dyes are studied by time-resolved fluorescence polarization anisotropy using intercalated ethidium as the probe. Existing theory is generalized to take account of excitation transfer between intercalated ethidiums, and Forster theory is shown to be valid in this context. The effects of intercalated ethidium, 9-aminoacridine, and proflavine on the torsional rigidity of linear and supercoiled DNAs are studied up to rather high binding ratios. Evidence is presented that metastable secondary structure persists in dye-relaxed supercoiled DNAs, which contradicts the standard model of supercoiled DNAs.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. A DNA minor groove electronegative potential genome map based on photo-chemical probing.

    PubMed

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten; Møllegaard, Niels Erik

    2011-08-01

    The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated, and it also appears that minor groove electronegative potential may contribute significantly in guiding proteins to their cognate binding sites in the genome. Based on the photo-chemical probing results, we have derived an algorithm that predicts the minor groove electronegative potential in a DNA helix of any given sequence. We have validated this model on a series of protein-DNA binding sites known to involve minor groove electrostatic recognition as well as on stable nucleosome core complexes. The algorithm allows for the first time a full minor groove electrostatic description at the nucleotide resolution of any genome, and it is illustrated how such detailed studies of this sequence dependent, inherent property of the DNA may reflect on genome organization, gene expression and chromosomal condensation.

  11. DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

    PubMed

    Rasche, Frank; Lueders, Tillmann; Schloter, Michael; Schaefer, Sabine; Buegger, Franz; Gattinger, Andreas; Hood-Nowotny, Rebecca C; Sessitsch, Angela

    2009-03-01

    A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

  12. Detection of foodborne pathogens using DNA probes and a dipstick format.

    PubMed

    Groody, E P

    1996-12-01

    The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such "gold standard" methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such as Salmonella, Listeria, and E. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.

  13. Local rheological probes for complex fluids: application to Laponite suspensions.

    PubMed

    Wilhelm, C; Elias, F; Browaeys, J; Ponton, A; Bacri, J-C

    2002-08-01

    We present an experimental method allowing a direct measurement of the local rheological behavior of complex fluids. A magnetic probe is inserted into the bulk of the fluid and submitted to a controlled magnetic force or torque, which induces a mechanical perturbation of the fluid. The geometry of the perturbation can be varied using two kinds of probes: a magnetic bead submitted to a homogeneous magnetic force in one direction, and a magnetic needle that can turn inside the material under the effect of an applied magnetic torque. Two complex viscoelastic fluids are investigated. First, a surfactant solution, which has a linear mechanical behavior in the range of the applied stresses, is used to test and validate the experimental methodology. We then use the local probes to investigate a Laponite colloidal suspension, which exhibits nonlinear behavior such as thixotropy, shear thinning, and aging. In this latter fluid, we find an exponential growth of the rheological relaxation time versus the system age, a power-law dependence of the fluid viscosity on the applied stress, and a dynamical yield stress which saturates with the fluid aging time.

  14. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

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

    PubMed

    Wyroba, E; Satir, B H

    2000-01-01

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

  16. 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.

  17. DNA-probe-target interaction based detection of Brucella melitensis by using surface plasmon resonance.

    PubMed

    Sikarwar, Bhavna; Singh, Virendra V; Sharma, Pushpendra K; Kumar, Ashu; Thavaselvam, Duraipandian; Boopathi, Mannan; Singh, Beer; Jaiswal, Yogesh K

    2017-01-15

    Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (KD) and maximum binding capacity of analyte (Bmax) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (KD) and 81.02m° (Bmax) with probe 1 and 54.9pM and 55.29m° (Bmax), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications.

  18. Evaluation of a prototype DNA probe test for the noncultural diagnosis of gonorrhea.

    PubMed Central

    Granato, P A; Franz, M R

    1989-01-01

    A prototype, nonisotopic, chemiluminescent DNA probe test called the Gen-Probe PACE (Probe Assay-Chemiluminescence Enhanced) system for Neisseria gonorrhoeae (Gen-Probe, San Diego, Calif.) was compared with conventional Martin-Lewis culture medium in JEMBEC plates for the laboratory diagnosis of gonorrhea. This 2-h noncultural assay is based upon the use of an acridinium ester-labeled DNA probe. The rRNA-directed DNA probe hybridizes with the target rRNA, and the hybridized probe is separated from the unhybridized probe through the use of magnetic microparticles. The esterified acridinium is hydrolyzed from the hybridized probe by the addition of an alkaline hydrogen peroxide solution, resulting in the production of visible light which is measured in a luminometer. The amount of light generated is directly proportional to the amount of gonococcal target rRNA present in the sample. A total of 407 clinical specimens (203 urethral and 204 endocervical) were collected from high-risk walk-in patients attending a sexually transmitted disease clinic. Separate patient specimens were collected for culture on Martin-Lewis medium in JEMBEC plates and for DNA probe assay. Statistical analysis of the overall comparative results showed that the DNA probe assay had a sensitivity, specificity, and positive and negative predictive values of 93, 99, 97, and 99%, respectively, in a patient population with a gonococcal disease prevalence of 21%. The results of this comparative study showed that the prototype chemiluminescent DNA probe assay is a rapid and reliable noncultural alternative for the laboratory diagnosis of gonorrhea. PMID:2498388

  19. 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.

  20. 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

  1. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  2. Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.

    PubMed

    Gollnick, Benjamin; Carrasco, Carolina; Zuttion, Francesca; Gilhooly, Neville S; Dillingham, Mark S; Moreno-Herrero, Fernando

    2015-03-18

    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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

    PubMed

    Guenther, Dale C; Anderson, Grace H; Karmakar, Saswata; Anderson, Brooke A; Didion, Bradley A; Guo, Wei; Verstegen, John P; Hrdlicka, Patrick J

    2015-08-01

    Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

  8. Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications†

    PubMed Central

    Guenther, Dale C.; Anderson, Grace H.; Karmakar, Saswata; Anderson, Brooke A.; Didion, Bradley A.; Guo, Wei; Verstegen, John P.; Hrdlicka, Patrick J.

    2015-01-01

    Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures – varying in the position, number, and distance between the intercalator zippers – are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology. PMID:26240741

  9. A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

    PubMed Central

    Glazer, A N; Peck, K; Mathies, R A

    1990-01-01

    The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble. Images PMID:2339125

  10. A stable double-stranded DNA-ethidium homodimer complex: Application to picogram fluorescence detection of DNA in agarose gels

    SciTech Connect

    Glazer, A.N.; Mathies, R.A. Lawrence Berkeley Laboratory, CA ); Peck, K. )

    1990-05-01

    The complex between double-stranded DNA and ethidium homodimer (5,5{prime}-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridinium) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the performed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

  11. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    PubMed

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.

  12. 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

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

    PubMed

    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.

  14. Study on the SPR responses of various DNA probe concentrations by parallel scan spectral SPR imaging

    NASA Astrophysics Data System (ADS)

    Ma, Suihua; Liu, Le; Lu, Weiping; Zhang, Yaou; He, Yonghong; Guo, Jihua

    2008-12-01

    SPR sensors have become a high sensitive and label free method for characterizing and quantifying chemical and biochemical interactions. However, the relations between the SPR refractive index response and the property (such as concentrations) of biochemical probes are still lacking. In this paper, an experimental study on the SPR responses of varies concentrations of Legionella pneumophila mip DNA probes is presented. We developed a novel two-dimensional SPR sensing technique-parallel scan spectral SPR imaging-to detect an array of mip gene probes. This technique offers quantitative refractive index information with a high sensing throughput. By detecting mip DNA probes with different concentrations, we obtained the relations between the SPR refractive index response and the concentrations of mip DNA probes. These results are valuable for design and developing SPR based mip gene biochips.

  15. 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.

  16. 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

  17. Energy-transfer-based wavelength-shifting DNA probes with "clickable" cyanine dyes.

    PubMed

    Holzhauser, Carolin; Rubner, Moritz M; Wagenknecht, Hans-Achim

    2013-05-01

    The insertion of cyanine dye azides as energy donor dyes via postsynthetic "click"-type cycloaddition chemistry with e.g. a new thiazole orange azide combined with thiazole red yields dual emitting DNA probes with good fluorescence readout properties.

  18. Computer programs used to aid in the selection of DNA hybridization probes.

    PubMed Central

    Raupach, R E

    1984-01-01

    This paper describes a package of three programs which used together aid in selecting the best possible sequence to be used as a DNA hybridization probe. This system searches an amino acid sequence for four adjacent amino acids with the fewest possible corresponding mRNA sequences, calculates their probability of occurrence, and locates the positions of wobbles and mismatches between the DNA hybridization probe and the possible mRNA sequences. PMID:6546442

  19. Release of DNA binary complexes from the ternary complexes by carboxymethyl poly(L-histidine).

    PubMed

    Asayama, Shoichiro; Sudo, Miyuki; Kawakami, Hiroyoshi

    2009-01-01

    The DNA ternary complexes with carboxymethyl poly(L-histidine) (CM-PLH) and poly(ethylenimine) (PEI) have released the DNA binary complexes with PEI by the protonation of CM-PLH at endosomal/lysosomal pH. The dissociation of the CM-PLH from the CM-PLH/PEI/DNA ternary complexes is proved by the fluorescence resonance energy transfer (FRET) analysis between the CM-PLH and PEI. The resulting PEI/DNA binary complexes easily released DNA, as compared with the CM-PLH/PEI/DNA ternary complexes, which was examined by competitive exchange with dextran sulfate. The release of the DNA binary complexes from the ternary complexes is promising mechanism for higher transfection activity by the CM-PLH/PEI/DNA ternary complexes.

  20. Streching of (DNA/functional molecules) complex between electrodes towards DNA molecular wire

    NASA Astrophysics Data System (ADS)

    Kobayashi, Norihisa; Nishizawa, Makoto; Inoue, Shintarou; Nakamura, Kazuki

    2009-08-01

    DNA/functional molecules such as (Ru(bpy)32+ complex, conducting polymer etc.) complex was prepared to study molecular structure and I-V characteristics towards DNA molecular wire. For example, Ru(bpy)32+ was associated with duplex of DNA by not only electrostatic interaction but also intercalation in the aqueous solution. Singlemolecular structure of DNA/Ru(bpy)32+ complex was analyzed with AFM. We found a network structure of DNA/Ru(bpy)32+ complex on the mica substrate, which is similar to native DNA. The height of DNA/Ru(bpy)32+ complex on the mica substrate was ranging from 0.8 to 1.6 nm, which was higher than the naked DNA (0.5-1.0 nm). This indicates that single-molecular DNA/Ru(bpy)32+ complex also connects to each other to form network structure on a mica substrate. In order to stretch DNA complex between electrodes, we employed high frequency and high electric field stretching method proposed by Washizu et al. We stretched and immobilized DNA single molecules between a pair of electrodes and its structures were analyzed with AFM technique. The I-V characteristics of DNA single molecules between electrodes were improved by the association of functional molecules with DNA. The molecular structure and I-V characteristics of DNA complex were discussed.

  1. Neutral red as a specific light-up fluorescent probe for i-motif DNA.

    PubMed

    Xu, Lijun; Wang, Jine; Sun, Na; Liu, Min; Cao, Yi; Wang, Zhili; Pei, Renjun

    2016-12-06

    We report a specific light-up fluorescent probe for i-motif DNA for the first time. Compared with the previously reported probes, neutral red could selectively interact with an i-motif and show a significant increase in its fluorescence. This feature makes it advantageous for designing label-free fluorescent sensing systems.

  2. Conformation of DNA in chromatin protein-DNA complexes studied by infrared spectroscopy.

    PubMed Central

    Liquier, J; Gadenne, M C; Taillandier, E; Defer, N; Favatier, F; Kruh, J

    1979-01-01

    The following observations concerning the DNA secondary structures in various nucleohistone complexes were made by infrared spectroscopy: 1/ in chromatin, chromatin extracted by 0.6 M NaCl, nucleosomes, and histone-DNA reconstituted complexes, the DNA remains in a B type conformation at low relative hygrometry; 2/ in chromatin extracted by tRNA and in non histone protein-DNA reconstituted complexes, the DNA can adopt an A type conformation. Infrared linear dichroism data show that in NHP-DNA complexes the low relative hygrometry conformation of DNA may be modified and that the infrared parameter -1090 is close to that measured for RNA's or DNA-RNA hybrids. It is concluded that the histones block the DNA in a B form and that some of the NHP could be involved in the control of the secondary structure of DNA in chromatin. Images PMID:450704

  3. 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.

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

    PubMed

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

    2015-03-02

    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.

  5. Employing double-stranded DNA probes on colloidal substrates for competitive hybridization events

    NASA Astrophysics Data System (ADS)

    Baker, Bryan Alexander

    DNA has found application beyond its biological function in the cell in a variety of materials assembly systems as well as nucleic acid-based detection devices. In the current research, double-stranded DNA probes are applied in both a colloidal particle assembly and fluorescent assay approach utilizing competitive hybridization interactions. The responsiveness of the double-stranded probes (dsProbes) was tuned by sequence design and tested against a variety of nucleic acid targets. Chapter 1 provides a review of the particle substrate used in the current research, colloidal particles, as well as examines previous applications of DNA in assembly and nucleic acid detection formats. Chapter 2 discusses the formation of fluorescent satellites, or similarly termed fluorescent micelles, via DNA hybridization. The effects of DNA duplex sequence, temperature at which assembly occurs, and oligonucleotide density are variables considered with preferential assembly observed for low oligonucleotide density particles. Chapter 3 demonstrates the controlled disassembly of these satellite structures via competitive hybridization with a soluble target strand. Chapter 4 examines DNA duplexes as fluorescent dsProbes and characterizes the kinetics of competitive hybridization between immobilized dsProbes and solution targets of interest. The sequence-based affinities of dsProbes as well as location of an embedded target sequence are both variables explored in this study. Based on the sequence design of the dsProbes, a range of kinetics responses are observed. Chapter 5 also examines the kinetics of competitive hybridization with dsProbes but with a focus on the specificity of competitive target by including mismatches within a short 15 base competitive target. Chapter 6 examines the effects of dsProbe orientation relative to the particle surface as well as substrate particle size. The kinetics of displacement of DNA targets with those of RNA targets of analogous sequence are also

  6. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  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.

  8. Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes.

    PubMed

    Xiang, Dong-shan; Zeng, Guo-ping; He, Zhi-ke

    2011-07-15

    We have developed a new analytical method to detect multiple DNA simultaneously based on the biobarcoded CdSe/ZnS quantum dot (QD) and magnetic microparticle (MMP). It was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus 16 and 18 L1 genes (HPV-16 and HPV-18) as model systems. This analytical system involves three types of probes, a MMP probe and two streptavidin-modified QD probes. The MMPs are functionalized with HPV-16 and HPV-18 captures DNA to form MMP probes. The QDs are conjugated with HPV-16 or HPV-18 probe DNA along with FAM- or Rox-labeled random DNA to form HPV-16 and HPV-18 QD probes, respectively. A one-step hybridization reaction was performed by mixing the MMP probes, HPV-16 and HPV-18 target DNA (T-16 and T-18), HPV-16 and HPV-18 QD probes. Afterwards, the hybrid-conjugated microparticles were separated by a magnet and heated to remove the MMPs. Finally, the detections of T-16 and T-18 were done by measuring fluorescence signals of FAM and Rox, respectively. Under the optimum conditions, the fluorescence intensity exhibited a good linear dependence on target DNA concentration in the range from 8 × 10⁻¹¹ to 8 × 10⁻⁹ M. The detection limit of T-16 is up to 7 × 10⁻¹¹ M (3σ), and that of T-18 is 6 × 10⁻¹¹ M. Compared with other biobarcode assay methods, the proposed method that QDs were used as the solid support has some advantages including shorter preparation time of QD probes, faster binding kinetics and shorter analytical time. Besides, it is simple and accurate.

  9. Hydrolytic cleavage of DNA by quercetin manganese(II) complexes.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-04-01

    Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 microM of the complexes is found to be 1.32 x 10(-4) s(-1). The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

  10. Probing the role of water in the tryptophan repressor-operator complex.

    PubMed Central

    Brown, M. P.; Grillo, A. O.; Boyer, M.; Royer, C. A.

    1999-01-01

    The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the

  11. 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.

  12. Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles.

    PubMed

    Zhang, Shusheng; Zhong, Hua; Ding, Caifeng

    2008-10-01

    A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.

  13. DNA interaction studies of a novel Cu(II) complex as an intercalator containing curcumin and bathophenanthroline ligands.

    PubMed

    Shahabadi, Nahid; Falsafi, Monireh; Moghadam, Neda Hosseinpour

    2013-05-05

    A new copper(II) complex; [Cu(Cur)(DIP)](+2) in which Cur=curcumin and DIP=4,7-diphenyl-1,10-phenanthroline, was synthesized and characterized using different physico-chemical methods. Binding interaction of this complex with calf thymus (CT-DNA) has been investigated by emission, absorption, circular dichroism, viscosity, and differential pulse voltammetry and fluorescence techniques. The complex displays significant binding properties to the CT-DNA. In fluorimeteric studies, the binding mode of the complex with CT-DNA was investigated using methylene blue as a fluorescence probe. Fluorescence of methylene blue-DNA solution increased in the presence of increasing amounts of the complex. It was found that the complex is able to displace the methylene blue completely. This indicate intercalation of the complex between base pairs of DNA. The cleavage of plasmid DNA by the complex was also studied. We found that the copper(II) complex can cleave puC18 DNA. Furthermore, mentioned complex induces detectable changes in the CD spectrum of CT-DNA, a decrease in absorption spectrum, and an increase in its viscosity. All of the experimental results showed that the Cu(II) complex bound to DNA by an intercalative mode of binding.

  14. 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.

  15. Ribosomal DNA spacer probes for yeast identification: studies in the genus Metschnikowia.

    PubMed

    Henriques, M; Sá-Nogueira, I; Giménez-Jurado, G; van Uden, N

    1991-02-01

    To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7.7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0.65 kb BamHI-HpaI fragment containing non-transcribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclusively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G + C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.

  16. Construction of specific DNA probe for the detection of Salmonella in food.

    PubMed

    Pilantanapak, A; Jayanetra, P; Panbangred, W; Klungthong, C; Bangtrakulnonth, A

    1997-03-01

    The Salmonella specific DNA fragment from genomic DNA of S. typhimurium ATCC 23566 was cloned in E. coli and successfully used as a digoxigenin labeled probe for detecting the presence of Salmonella serotypes in both artificially contaminated food and natural contaminated food samples.

  17. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    PubMed

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  18. Generic technique to generate large branched DNA complexes.

    PubMed

    Tosch, Paul; Wälti, Christoph; Middelberg, Anton P J; Davies, A Giles

    2006-03-01

    The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

  19. 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.

  20. A DNA probe based on phosphorescent resonance energy transfer for detection of transgenic 35S promoter DNA.

    PubMed

    Lv, Jinzhi; Miao, Yanming; Yang, Jiajia; Qin, Jin; Li, Dongxia; Yan, Guiqin

    2017-05-15

    A QDs-DNA nano-probe was made by combining Mn-doped ZnS room-temperature phosphorescence (RTP) quantum dots (QDs) and DNA. Then an RTP sensor for quantitative detection of genetically-modified mark sequence cauliflower mosaic virus 35S promoter (Ca MV 35S) DNA was built on basis of phosphorescent resonance energy transfer (PRET). The underlying principles were that a QDs-DNA water-soluble nano-probe was built by connecting single-strand DNA to the surfaces of QDs via a ligand exchange method. This probe had good RTP performance and could well identify Ca MV 35S. Thereby, the simple, rapid and efficient detection of genetically-modified organisms was realized. With the increase of target DNA sequence, the phosphorescent intensity of QDs was gradually reduced due to the energy transfer between QDs and the organic quencher BHQ2. This sensor had a detection limit of 4.03nM and a detection range of 12-300nM. Moreover, this sensor had high selectivity. This sensor could effectively detect the target DNA compared with mismatched and random sequences. Thus, this method is very promising for biological analysis.

  1. Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

    PubMed

    Tous, G; Fausnaugh, J; Vieira, P; Stein, S

    1988-07-01

    A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

  2. Macrocyclic Metal Complex-DNA Conjugates for Electrochemical Sensing of Single Nucleobase Changes in DNA.

    PubMed

    Duprey, Jean-Louis H A; Carr-Smith, James; Horswell, Sarah L; Kowalski, Jarosław; Tucker, James H R

    2016-01-27

    The direct incorporation of macrocyclic cyclidene complexes into DNA via automated synthesis results in a new family of metal-functionalized DNA derivatives that readily demonstrate their utility through the ability of one redox-active copper(II)-containing strand to distinguish electrochemically between all four canonical DNA nucleobases at a single site within a target sequence of DNA.

  3. A new system for the amplification of biological signals: RecA and complimentary single strand DNA probes on a leaky surface acoustic wave biosensor.

    PubMed

    Zhang, Liqun; Wang, Yunxia; Chen, Ming; Luo, Yang; Deng, Kun; Chen, Dong; Fu, Weiling

    2014-10-15

    This research describes a new amplification signals system of the leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor for the simple, sensitive and rapid detection of the target double-stranded DNA (dsDNA). The system consists of a RecA protein-coated complementary single-stranded DNA (cssDNA) probe complex that amplifies the biological signal to improve the sensitivity of the biosensor. The bis-PNA probe for detecting HPV was first immobilized on a gold surface membrane of the detection channel. After the probe was completely hybridized with the corresponding target DNA, different concentrations of the "RecA protein-complementary single strand DNA probe" were added to react with the bis-PNA/dsDNA complex. The phase shift of the LSAW biosensors, which was measured and found to be most significant when the RecA protein was 45 μg/mL and the ATPγS was 2.5 mmol/L. Compared with other concentrations (P<0.01) of RecA and ATPγS, the value of the phase shift was (11.74 ± 1.03) degrees and the ratio of the phase shift and hybridization time clearly outperformed that of the other concentrations. Compared to the direct hybridization of the bis-PNA probe and the target DNA sequence, the sensitivity was effectively improved and the detection time was significantly shortened. PNA binding adjacent to the area of the target sequence homologous to the probe significantly increased the yield of the hybridization reaction between the PNA/dsDNA complex and the RecA protein-coated cssDNA probe. In this condition, the phase shift was significantly obvious and the detection time was significantly shortened. In conclusion, the combination of the RecA protein-coated cssDNA probe and the LSAW bis-PNA biosensor provides sensitivity and simple and rapid detection of clinical trace pathogenic microorganisms.

  4. Complex DNA nanostructures from oligonucleotide ensembles.

    PubMed

    Mathur, Divita; Henderson, Eric R

    2013-04-19

    The first synthetic DNA nanostructures were created by self-assembly of a small number of oligonucleotides. Introduction of the DNA origami method provided a new paradigm for designing and creating two- and three-dimensional DNA nanostructures by folding a large single-stranded DNA and 'stapling' it together with a library of oligonucleotides. Despite its power and wide-ranging implementation, the DNA origami technique suffers from some limitations. Foremost among these is the limited number of useful single-stranded scaffolds of biological origin. This report describes a new approach to creating large DNA nanostructures exclusively from synthetic oligonucleotides. The essence of this approach is to replace the single-stranded scaffold in DNA origami with a library of oligonucleotides termed "scaples" (scaffold staples). Scaples eliminate the need for scaffolds of biological origin and create new opportunities for producing larger and more diverse DNA nanostructures as well as simultaneous assembly of distinct structures in a "single-pot" reaction.

  5. Probing the complex ionic structure of warm dense carbon

    NASA Astrophysics Data System (ADS)

    Kraus, Dominik

    2014-10-01

    The carbon phase diagram at extreme pressure conditions has received broad interest for modeling planetary interiors and high energy density laboratory experiments. Numerous theoretical models and simulations have recently been performed but critical experimental data at the phase boundaries and of the microscopic physical properties remain very scarce. In this work, we present novel experimental observations of the complex ion structure in warm dense carbon at pressures from 20 to 220 GPa and temperatures of several thousand Kelvins. Our experiments employ powerful x-ray sources at kilo-joule class laser facilities and at the Linac Coherent Light Source to perform spectrally and angularly resolved x-ray scattering from shock-compressed graphite samples; the absolute static ion structure factor is directly measured by resolving the ratio of elastically and inelastically scattered radiation. Using different types of graphite and varying drive laser intensity, we were able to probe conditions below and above the melting line, resolving the shock-induced graphite-to-diamond and graphite-to-liquid transitions on nanosecond time scale. Our results confirm a complex ionic structure predicted by QMD simulations and demonstrate the importance of chemical bonds at extreme conditions similar to those found in the interiors of giant planets. The evidence presented here thus provides a firmer ground for modeling the evolution and current structure of carbon-bearing icy giants like Neptune, Uranus, and a number of extra-solar planets.

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

    PubMed

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

    2005-01-01

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

  7. Probing DNA by 2-OG-dependent dioxygenase

    PubMed Central

    Tsai, Chi-Lin; Tainer, John A.

    2014-01-01

    TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation. PMID:24360270

  8. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    PubMed

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  9. Hydrolytic cleavage of DNA by quercetin zinc(II) complex.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-03-01

    Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

  10. 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.

  11. Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

    PubMed Central

    Nagy, E

    1992-01-01

    A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1316798

  12. Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

    PubMed Central

    Edgcomb, Virginia P.; McDonald, John H.; Devereux, Richard; Smith, David W.

    1999-01-01

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 107 to 2.5 × 109 (average, 1.1 × 109 ± 5.2 × 108) cells g of sediment−1. In September, numbers of SRB detected ranged from 5.4 × 108 to 7.3 × 109 (average, 2.5 × 109 ± 1.5 × 109) cells g of sediment−1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments. PMID:10103245

  13. 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.

  14. Disruption of a Topoisomerase-DNA Cleavage Complex by a DNA Helicase

    NASA Astrophysics Data System (ADS)

    Howard, Michael T.; Neece, Sue H.; Matson, Steven W.; Kreuzer, Kenneth N.

    1994-12-01

    The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  16. Structure of Escherichia coli AlkA in Complex with Undamaged DNA*

    PubMed Central

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

    2010-01-01

    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. PMID:20843803

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

    DOE PAGES

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; ...

    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 withmore » 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.« less

  18. Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

    PubMed Central

    Darrasse, A; Kotoujansky, A; Bertheau, Y

    1994-01-01

    Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed. Images PMID:8117082

  19. 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.

  20. 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.

  1. Studies on the Interaction Mechanism of 1,10-Phenanthroline Cobalt(II) Complex with DNA and Preparation of Electrochemical DNA Biosensor

    PubMed Central

    Niu, Shuyan; Li, Feng; Zhang, Shusheng; Wang, Long; Li, Xuemei; Wang, Shiying

    2006-01-01

    Fluorescence spectroscopy and ultraviolet (UV) spectroscopy techniques coupled with cyclic voltammetry (CV) were used to study the interaction between salmon sperm DNA and 1,10-Phenanthroline cobalt(II) complex, [Co(phen)2(Cl)(H2O)]Cl·H2O, where phen = 1,10-phenanthroline. The interaction between [Co(phen)2(Cl)(H2O)]+ and double-strand DNA (dsDNA) was identified to be intercalative mode. An electrochemical DNA biosensor was developed by covalent immobilization of probe single-strand DNA (ssDNA) related to human immunodeficiency virus (HIV) on the activated glassy carbon electrode (GCE). With [Co(phen)2(Cl)(H2O)]+ being the novel electrochemical hybridization indicator, the selectivity of ssDNA-modified electrode was investigated and selective detection of complementary ssDNA was achieved using differential pulse voltammetry (DPV).

  2. 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

    2013-12-03

    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

  3. Use of DNA Probes for Diagnosis of Infectious Diseases.

    DTIC Science & Technology

    1987-01-01

    cloned D sequences and mre recently clinical and enviromental sales. The fundamental ongets behind nuc -leic acid inr** m~licatigos. Nucleic acid probe...Professor of Pldical icrobiology and Virology In the School of Public Health and is Director of the Naval Biosciences Laboratory. The research reported here was supported by the Office of Naval Research. - ~-w~ -

  4. A method for recovering strand-specific probes from nick-translated DNA fragments.

    PubMed

    Dutton, F L; Chovnick, A

    1984-07-01

    A method of preparing strand-specific probes for DNA X DNA or DNA X RNA hybridizations is described. Double-stranded DNA fragments are first isolated from any recombinant DNA clone containing the desired sequence, and then labeled in vitro by nick-translation (T. Maniatis, A. Jeffrey, and D. G. Kleid (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188; P. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg (1977) J. Mol. Biol. 113, 237-251). Sequences homologous to the desired strand are captured by annealing the denatured nick-translate to viral strands of an appropriate M13 clone, and recovered by elution of the resulting hybrids from a column of agarose A50M (Bio-Rad). By this method, separate probes with specificity to either strand, as well as the double-stranded probe, may conveniently be prepared from a single nick-translation reaction. Probes may be obtained which are homologous either to the full length of the cloned region or to selected portions thereof by selecting appropriate M13 clones for annealing. The probe is recovered as a population of fragments several hundred bases or less in length, which have been found ideal for saturating liquid hybridizations, and should be similarly well suited for in situ hybridizations to cytological preparations.

  5. Effect of different 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.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    This paper principally delineates to the detection process of Human Papillomavirus (HPV) DNA test. HPV is an extremely common virus infection that infected to human by the progressions cell in the cervix cell. The types of HPV that give a most exceedingly awful infected with cervical cancer is 16 and 18 other than 31 and 45. The HPV DNA probe is immobilized with a different concentration to stabilize the sensitivity. A technique of rapid and sensitive for the HPV identification was proposed by coordinating basic DNA extraction with a quality of DNA. The extraction of the quality of DNA will make a proficiency of the discovery procedure. It will rely on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization procedures are described by current-voltage (I-V) estimation by utilizing KEITHLEY 6487. This procedure will play out a decent affectability and selectivity of HPV discovery.

  6. Effect of structure on sensing performance of a target induced signaling probe shifting DNA-based (TISPS-DNA) sensor.

    PubMed

    Yu, Xiang; Yu, Zhigang; Li, Fengqin; Xu, Yanmei; He, Xunjun; Xu, Lan; Shi, Wenbing; Zhang, Guiling; Yan, Hong

    2017-05-15

    A type of "signal on" displacement-based sensors named target induced signaling probe shifting DNA-based (TISPS-DNA) sensor were developed for a designated DNA detection. The signaling mechanism of the signaling probe (SP) shifting different from the classical conformation/flexibility change mode endows the sensor with high sensitivity. Through using thiolated or no thiolated capturing probe (CP), two 3-probe sensing structures, sensor-1 and sensor-2, were designed and constructed. The systematical comparing research results show that both sensors exhibit some similarities or big differences in sensing performance. On the one hand, the similarity in structures determines the similarity in some aspects of signaling mechanism, background signal, signal changing form, anti-fouling ability and versatility; on the other hand, the slight difference in structures also results in two opposite hybridization modes of gradual increasing resistance and gradual decreasing resistance which can affect the hybridization efficiency between the assistant probe (AP) and the SP, further producing some big differences in sensing performance, for example, apparently different signal enhancement (SE) change, point mutation discrimination ability and response speed. Under the optimized fabrication and detection conditions, both sensors feature high sensitivity for target DNAs with the detection limits of ∼10 fM for sensor-1 and ∼7 fM for sensor-2, respectively. Among many acquired sensing virtues, the sensor-1 shows a peculiar specificity adjustability which is also a highlight in this work.

  7. 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.

  8. Force-extension relation of DNA-histone complexes

    NASA Astrophysics Data System (ADS)

    Levine, A. J.; Henle, Mark L.; Chou, Tom

    2007-03-01

    In eukaryotic cells, DNA is packaged inside the nucleus in the form of chromatin, a structure whose basic repeat unit, known as the nucleosome, consists of DNA wrapped around a cylindrical complex of histone proteins. In order for the cell to function properly, these nucleosome complexes must be stable at equilibrium. At the same time, the cell must be able to gain access to the genomic information contained within the DNA, which it can achieve by exerting forces on the nucleosomes that cause the DNA to unwrap from the histones. Single molecule mechanical manipulation techniques, in which DNA/histone complexes are disrupted by an external force, can provide information not only about the equilibrium structure of these complexes, but also about the forces and displacements required to access the DNA in the nucleosome. In this talk, we derive the force-extension relation for these complexes. We allow for the DNA to unwrap from the histones in both a continuous and discontinuous fashion; that is, we allow the histones to ``pop'' off of the DNA, releasing a large amount of DNA in the process. We also include the conformational fluctuations of the unwrapped portions of the DNA.

  9. Metal Complexes for DNA-Mediated Charge Transport

    PubMed Central

    Barton, Jacqueline K.; Olmon, Eric D.; Sontz, Pamela A.

    2010-01-01

    In all organisms, oxidation threatens the integrity of the genome. DNA-mediated charge transport (CT) may play an important role in the generation and repair of this oxidative damage. In studies involving long-range CT from intercalating Ru and Rh complexes to 5′-GG-3′ sites, we have examined the efficiency of CT as a function of distance, temperature, and the electronic coupling of metal oxidants bound to the base stack. Most striking is the shallow distance dependence and the sensitivity of DNA CT to how the metal complexes are stacked in the helix. Experiments with cyclopropylamine-modified bases have revealed that charge occupation occurs at all sites along the bridge. Using Ir complexes, we have seen that the process of DNA-mediated reduction is very similar to that of DNA-mediated oxidation. Studies involving metalloproteins have, furthermore, shown that their redox activity is DNA-dependent and can be DNA-mediated. Long range DNA-mediated CT can facilitate the oxidation of DNA-bound base excision repair proteins to initiate a redox-active search for DNA lesions. DNA CT can also activate the transcription factor SoxR, triggering a cellular response to oxidative stress. Indeed, these studies show that within the cell, redox-active proteins may utilize the same chemistry as that of synthetic metal complexes in vitro, and these proteins may harness DNA-mediated CT to reduce damage to the genome and regulate cellular processes. PMID:21643528

  10. 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.

  11. Hydrodynamic properties of DNA and DNA-lipid complex in an elongational flow field.

    PubMed

    Sasaki, Naoki; Ashitaka, Hidetomo; Ohtomo, Kenji; Fukui, Akimasa

    2007-03-10

    The aim of this study was to determine the difference between hydrodynamic properties of DNA-cetyltrimethylammonium (CTA) complex and those of DNA, which may be related to the difference in fibre-forming ability of DNA-CTA from that of DNA. Responses of DNA and DNA-CTA complex to an elongational flow field were investigated. In both solution systems, results suggesting a coil-stretch transition were obtained. From a critical strain rate value, the radius of gyration of DNA-CTA molecules in ethanol-glycerol solution was revealed to be 0.3-0.5 times of that of DNA in aqueous NaCl solution. Shear viscosity of DNA-CTA solution was much smaller than that of DNA solution, also suggesting a smaller size of DNA-CTA in ethanol-glycerol solution than that of DNA in aqueous NaCl solution. The plateau birefringence value of the DNA-CTA system, a parameter that indicates the local molecular conformation and the molecular arrangement, was only about 1/10 of that of the DNA system. There is an empirically determined molecular model of DNA-CTA complex in which a DNA molecule is sheathed by a cylindrical crust made of CTA chains. This structure reduces the DNA molecular density in a pure elongational flow field region but cannot explain the observed reduction of birefringence intensity. The small plateau birefringence value of DNA-CTA compared with that of DNA was attributed to the reduced molecular polarizability by the particular conformation of DNA molecules and CTA chains in the DNA-CTA system such as that expected by the conformational models.

  12. Two-photon AgNP/DNA-TP dye nanosensing conjugate for biothiol probing in live cells.

    PubMed

    Liu, Mingli; Tang, Qiao; Deng, Ting; Yan, Huijuan; Li, Jishan; Li, Yinhui; Yang, Ronghua

    2014-12-07

    A novel silver nanoparticle (AgNP)/DNA-two-photon dye (TP dye) conjugate was fabricated as a two-photon nanoprobe for biothiol imaging in live cells. DNA-templated silver nanoparticles are efficient quenchers and also provide a biocompatible nanoplatform for facile delivery of DNA into living cells. In the presence of biothiols (Cys, Hcy, or GSH), the strong interaction between the thiol group and silver results in the release of TP dye-labeled single-stranded DNA (ssDNA) from the AgNP surface and the subsequent fluorescence emission of the TP dye, thus enabling biothiols to be assayed. Our results reveal that the AgNP/DNA-TP dye nanosensing conjugate not only is a robust, sensitive, and selective sensor for quantitative detection of biothiols in the complex biological environment but also can be efficiently delivered into live cells and act as a "signal-on" sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology for the development of future DNA-templated silver nanoparticle-based two-photon fluorescent probes for use in vitro or in vivo as biomolecular sensors for live-cell imaging.

  13. 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.

  14. Ultrafast fluorescence dynamics of Sybr Green I/DNA complexes

    NASA Astrophysics Data System (ADS)

    Trantakis, Ioannis A.; Fakis, Mihalis; Tragoulias, Sotirios S.; Christopoulos, Theodore K.; Persephonis, Peter; Giannetas, Vassilis; Ioannou, Penelope

    2010-01-01

    The ultrafast dynamics of the DNA fluorescent dye Sybr Green I (SG) has been studied in buffer, single-stranded (ssDNA), double-stranded (dsDNA) and triple-stranded DNA (tsDNA). The fluorescence quantum yield of SG increases dramatically when bound to DNA (including tsDNA). The fluorescence dynamics of the free SG has shown two decay components with ˜0.15-0.4 ps and ˜1.3-2.1 ps time constants, depending on the fluorescence wavelength. Upon binding to DNA, the dynamics becomes slower exhibiting four decay components. This is mainly due to the restriction of the internal motions of the dye caused by the relatively rigid environment of the dye complexed with DNA.

  15. Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

    PubMed

    Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

    2016-12-15

    In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM.

  16. Evolution of a complex minisatellite DNA sequence.

    PubMed

    Barros, Paula; Blanco, Miguel G; Boán, Francisco; Gómez-Márquez, Jaime

    2008-11-01

    Minisatellites are tandem repeats of short DNA units widely distributed in genomes. However, the information on their dynamics in a phylogenetic context is very limited. Here we have studied the organization of the MsH43 locus in several species of primates and from these data we have reconstructed the evolutionary history of this complex minisatellite. Overall, with the exception of gibbon, MsH43 has an organization that is asymmetric, since the distribution of repeats is distinct between the 5' and 3' halves, and heterogeneous since there are many different repeats, some of them characteristic of each species. Inspection of the MsH43 arrays showed the existence of many duplications and deletions, suggesting the implication of slippage processes in the generation of polymorphism. Concerning the evolutionary history of this minisatellite, we propose that the birth of MsH43 may be situated before the divergence of Old World Monkeys since we found the existence of some MsH43 repeat motifs in prosimians and New World Monkeys. The analysis of MsH43 in apes revealed the existence of an evolutionary breakpoint in the pathway that originated African great apes and humans. Remarkably, human MsH43 is more homologous to orang-utan than to the corresponding sequence in gorilla and chimpanzee. This finding does not comply with the evolutionary paradigm that continuous alterations occur during the course of genome evolution. To adjust our results to the standard phylogeny of primates, we propose the existence of a wandering allele that was maintained almost unaltered during the period that extends between orang-utan and humans.

  17. Identification of Streptococcus pneumoniae with a DNA probe.

    PubMed Central

    Denys, G A; Carey, R B

    1992-01-01

    The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae. PMID:1400974

  18. 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.

  19. Coaxial atomic force microscope probes for dielectrophoresis of DNA under different buffer conditions

    NASA Astrophysics Data System (ADS)

    Tao, Yinglei; Kumar Wickramasinghe, H.

    2017-02-01

    We demonstrate a coaxial AFM nanoprobe device for dielectrophoretic (DEP) trapping of DNA molecules in Tris-EDTA (TE) and phosphate-buffered saline (PBS) buffers. The DEP properties of 20 nm polystyrene beads were studied with coaxial probes in media with different conductivities. Due to the special geometry of our DEP probe device, sufficiently high electric fields were generated at the probe end to focus DNA molecules with positive DEP. DEP trapping for both polystyrene beads and DNA molecules was quantitatively analyzed over the frequency range from 100 kHz to 50 MHz and compared with the Clausius-Mossotti theory. Finally, we discussed the negative effect of medium salinity during DEP trapping.

  20. Synthetic exfoliative toxin A and B DNA probes for detection of toxigenic Staphylococcus aureus strains.

    PubMed Central

    Rifai, S; Barbancon, V; Prevost, G; Piemont, Y

    1989-01-01

    Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in electrosyneresis harbored the ET gene(s). The latter strains produced levels of ET. ET-negative phage group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s). The sensitivity of the DNA hybridization technique was 10(6) bacteria or 100 ng of genomic DNA. Images PMID:2715322

  1. A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes.

    PubMed

    Sato, Norihiro; Tsuji, Genichiro; Sasaki, Yoshihiro; Usami, Akira; Moki, Takuma; Onizuka, Kazumitsu; Yamada, Ken; Nagatsugi, Fumi

    2015-10-14

    Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst-AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

  2. Probing the linearity and nonlinearity in DNA sequences

    NASA Astrophysics Data System (ADS)

    Tsonis, Anastasios A.; Heller, Fred L.; Tsonis, Panagiotis A.

    2002-09-01

    In this paper, we apply the principles of information theory that relate to the definition of nonlinear predictability, which is a measure that describes both the linear and nonlinear components of a system. By comparing this measure to a measure of linear predictability, one can assess whether a given system has a strong linear or a strong nonlinear component. This provides insights as to whether the system should be modeled by a nonlinear or a linear model. We apply these ideas to DNA sequences. Our results, which extend previous results on this issue indicate that all DNA sequences (coding and noncoding) exhibit strong nonlinear structure. At the same time the results provide insights to understand DNA structure and possible clues about evolutionary mechanisms.

  3. 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.

  4. Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes

    SciTech Connect

    Phoebe L. Stewart

    2005-06-27

    Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second

  5. Clinical application of novel sample processing technology for the identification of salmonellae by using DNA probes.

    PubMed

    Scholl, D R; Kaufmann, C; Jollick, J D; York, C K; Goodrum, G R; Charache, P

    1990-02-01

    Two hundred and fifty clinical fecal specimens collected over a 7-month period were analyzed for the presence of salmonellae by a rapid DNA hybridization procedure. Hybridizations were performed by using a novel specimen processing protocol called wicking and a previously unreported 1,600-base-pair probe cloned from Salmonella enteritidis DNA. The probe was shown to be reactive with all 70 Salmonella serotypes tested and not reactive with 101 stock strains of other enteric bacteria. Southern analysis of 30 Salmonella isolates representing 22 serotypes suggested that the probe sequence was highly conserved, appearing as a 1,600-base-pair band in a BglII digest of isolate DNA in 29 of 30 isolates and as a 2,300-base-pair fragment in 1 of the isolates. The probe correctly identified all salmonellae (nine isolates) among 47 H2S-producing colonies tested from among 250 clinical specimens cultured on xylose-lysine-desoxycholate medium. Salmonellae grown on xylose-lysine-desoxycholate medium gave consistently higher hybridization values than did those grown on either MacConkey or Hektoen enteric agar. In addition, of eight gram-negative broth enrichments in which salmonellae were identified by conventional means, seven were probe positive. The use of this nucleic acid probe and hybridization technique provides a simple and rapid identification of Salmonella species.

  6. 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

  7. Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

    PubMed Central

    Wu, Jun; Shao, Fangwei; Zhang, Li-Feng

    2014-01-01

    Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts. PMID:25226542

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

    PubMed

    Welling, Ryan C; Knotts, Thomas A

    2015-01-07

    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.

  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. 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.

  11. 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.

  12. New naphthalimide modified polyethylenimine nanoparticles as fluorescent probe for DNA detection

    NASA Astrophysics Data System (ADS)

    Liang, Shucai; Yu, Hui; Xiang, Jin; Yang, Wang; Chen, Xiaohui; Liu, Yanbin; Gao, Chen; Yan, Guoping

    2012-11-01

    A new naphthalimide modified polyethylenimine (PEI) nanoparticles (called NPEI-NPs) was synthesized and applied as fluorescent probe for rapid, selective and sensitive fluorometric detection of trace DNA. The synthesis involved the covalent modification of PEI with 4-butylamino-N-carboxymethyl-1,8-naphthalimide(BACMN) for getting amphiphilic polymer. Then the amphiphilic polymer was self-assembled in water to give the NPEI-NPs. NPEI-NPs was soluble in water and emitted fluorescence at 545 nm with exciting at 460 nm. The fluorescence spectra resulting from the interaction between NPEI-NPs and DNA indicated that the fluorescence of NPEI-NPs increased in the present of DNA. Therefore, a fluorescence enhancement method was developed for the determination of trace fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA). Under the optimal conditions, the calibration curves were linear over the concentration ranges of 0.05-2.8 μg/mL for fsDNA and 0.08-3.0 μg/mL for ctDNA. The detection limits for fsDNA and ctDNA were 1.6 and 2.0 ng/mL, respectively. The proposed method has been employed to quantify DNA in synthetic samples with the satisfactory results.

  13. Threading of Binuclear Ruthenium Complex Through DNA Bases

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan; Westerlund, Fredrik; McCauley, Micah; Lincoln, Per; Rouzina, Ioulia; Williams, Mark

    2009-03-01

    Due to steric constraints the dumb-bell shaped binuclear ruthenium complex can only intercalate DNA by threading, which requires local melting of the DNA to occur. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to threading compared to bulk experiments. Stretching single DNA molecules with different drug concentrations and holding a constant force allows the binding to reach equilibrium. We can obtain the equilibrium fractional ligand binding and length of DNA at saturation. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics. In addition, we obtain the minimum binding site size, which may be determined by either electrostatic repulsion or steric constraints.

  14. DNA complexes with Ni nanoparticles: structural and functional properties

    NASA Astrophysics Data System (ADS)

    Tatarinova, Olga N.; Smirnov, Igor P.; Safenkova, Irina V.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2012-10-01

    Supramolecular complexes of biopolymers based on magnetic nanoparticles play an important role in creation of biosensors, implementation of theragnostic and gene therapeutic methods and biosafety evaluation. We investigated the impact of DNA interactions with nanoparticles of nickel (nNi) on the integrity and functionality of DNA. Data obtained by mass spectrometry, electrophoresis, TEM and AFM microscopy techniques, bacterial transformation, and real-time PCR provide evidence that ssDNA and plasmid DNA (pDNA) efficiently form complexes with nNi. AFM data suggest that the complexes are necklace-type structures, in which nanoparticles are randomly distributed along the DNA chains, rather than highly entangled clot-type structures. After desorption, observed DNA characteristics in bioanalytical and biological systems remain unchanged. Only supercoiled pDNA was nicked, but remained, as well as a plasmid-nNi complex, active in expression vector assays. These results are very important for creation of new methods of DNA immobilization and controlled manipulation.

  15. 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

  16. 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

    2014-05-02

    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.

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

    PubMed

    Wu, Yao; Lai, Rebecca Y

    2014-09-02

    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.

  18. 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

  19. Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.

    PubMed

    Schneider, Nils; Meier, Matthias

    2017-02-01

    Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications.

  20. Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.

    PubMed

    Aranda, E; Rodríguez, M; Benito, M J; Asensio, M A; Córdoba, J J

    2002-06-05

    A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.

  1. General method for cloning amplified DNA by differential screening with genomic probes.

    PubMed Central

    Brison, O; Ardeshir, F; Stark, G R

    1982-01-01

    Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or

  2. 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.

  3. Quantum interference in DNA bases probed by graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Jeong, Heejeong; Seul Kim, Han; Lee, Sung-Hoon; Lee, Dongho; Hoon Kim, Yong; Huh, Nam

    2013-07-01

    Based on first-principles nonequilibrium Green's function calculations, we demonstrate quantum interference (QI) effects on the tunneling conductance of deoxyribonucleic acid bases placed between zigzag graphene nanoribbon electrodes. With the analogy of QI in hydrocarbon ring structures, we hypothesize that QI can be well preserved in the π-π coupling between the carbon-based electrode and a single DNA base. We demonstrate indications of QI, such as destructively interfered anti-resonance or Fano-resonance, that affect the variation of tunneling conductance depending on the orientation of a base. We find that guanine, with a 10-fold higher transverse conductance, can be singled out from the other bases.

  4. 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.

  5. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    PubMed

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  6. 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.

  7. 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

  8. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

    PubMed Central

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. PMID:27369379

  9. DNA-Based MRI Probes for Specific Detection of Chronic Exposure to Amphetamine in Living Brains

    PubMed Central

    Liu, Christina H.; Ren, Jia Q.; Yang, Jinsheng; Liu, Charng-ming; Mandeville, Joseph B.; Rosen, Bruce R.; Bhide, Pradeep G.; Yanagawa, Yuchio; Liu, Philip K.

    2009-01-01

    We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and ΔfosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ΔfosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled ΔfosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ΔfosB mRNA in living brains. PMID:19710318

  10. Development of a biotinylated DNA probe for detection of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Deering, R.E.; Arakawa, C.K.; Oshima, K.H.; O'Hara, P.J.; Landolt, M.L.; Winton, J.R.

    1991-01-01

    A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \

  11. Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles.

    PubMed

    Guenther, Dale C; Karmakar, Saswata; Hrdlicka, Patrick J

    2015-10-18

    Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.

  12. Cyanines as new fluorescent probes for DNA detection and two-photon excited bioimaging.

    PubMed

    Feng, Xin Jiang; Wu, Po Lam; Bolze, Frédéric; Leung, Heidi W C; Li, King Fai; Mak, Nai Ki; Kwong, Daniel W J; Nicoud, Jean-François; Cheah, Kok Wai; Wong, Man Shing

    2010-05-21

    A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.

  13. 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

  14. Local probe investigation of emergent phenomena in complex oxide heterointerfaces

    NASA Astrophysics Data System (ADS)

    Huang, Mengchen

    Complex oxide heterointerfaces exhibit rich physics as well as many veiled puzzles. LaAlO3/SrTiO3 (LAO/STO) is one of the prototype of such heterointerfaces. In 2004, Ohtomo and Hwang first reported a conducing interface emerged between perovskite oxide insulators LaAlO3 and SrTiO3. Following this seminal discovery, many emergent phenomena like metal-insulator transition, piezoresponse, superconductivity, magnetism, strong spin-orbit coupling and coexistence of superconductivity and magnetism were reported in the fascinating LAO/STO system. However, the origin of the conducting interface is still the subject of intense debate, and the physics behind these emergent phenomena remains a wild space to be explored. My Ph.D. study focused on the emergent phenomena in LAO/STO by using "local probes" -- nanostructures created by conductive atomic force microscope (c-AFM) lithography and the AFM itself. I used piezoresponse force microscope (PFM) to study the electromechanical response in LAO/STO and developed a high-resolution, non-destructive PFM imaging technique to visualize nanostructures at LAO/STO interface. The results indicate that the PFM signal is related to a carrier density mediated interfacial lattice distortion, and surface adsorbates can affect the PFM signal via coupling to the electrons at the interface. I integrated graphene on LAO/STO, created field-effect devices in graphene/LAO/STO and collaborated with Dr. Giriraj Jnawali to investigate the transport properties. The high quality single layer graphene on LAO/STO exhibited the half-integer quantum Hall effect and room temperature weak antilocalization behavior. I performed transport measurements in (110)-oriented LAO/STO to investigate anisotropic quasi one-dimensional superconductivity in nanowires. Based on the results I proposed a plausible explanation related to the Lifshitz transition and anisotropic band structures of nanowires in (110)-oriented LAO/STO. Co-worked with Dr. Keith Brown, I studied

  15. Scanning probe microscopy investigation of complex-oxide heterostructures

    NASA Astrophysics Data System (ADS)

    Bi, Feng

    Advances in the growth of precisely tailored complex-oxide heterostructures have led to new emergent behavior and associated discoveries. One of the most successful examples consists of an ultrathin layer of LaAlO 3 (LAO) deposited on TiO2-terminated SrTiO3 (STO), where a high mobility quasi-two dimensional electron liquid (2DEL) is formed at the interface. Such 2DEL demonstrates a variety of novel properties, including field tunable metal-insulator transition, superconductivity, strong spin-orbit coupling, magnetic and ferroelectric like behavior. Particularly, for 3-unit-cell (3 u.c.) LAO/STO heterostructures, it was demonstrated that a conductive atomic force microscope (c-AFM) tip can be used to "write" or "erase" nanoscale conducting channels at the interface, making LAO/STO a highly flexible platform to fabricate novel nanoelectronics. This thesis is focused on scanning probe microscopy studies of LAO/STO properties. We investigate the mechanism of c-AFM lithography over 3 u.c. LAO/STO in controlled ambient conditions by using a vacuum AFM, and find that the water molecules dissociated on the LAO surface play a critical role during the c-AFM lithography process. We also perform electro-mechanical response measurements over top-gated LAO/STO devices. Simultaneous piezoresponse force microscopy (PFM) and capacitance measurements reveal a correlation between LAO lattice distortion and interfacial carrier density, which suggests that PFM could not only serve as a powerful tool to map the carrier density at the interface but also provide insight into previously reported frequency dependence of capacitance enhancement of top-gated LAO/STO structures. To study magnetism at the LAO/STO interface, magnetic force microscopy (MFM) and magnetoelectric force microscopy (MeFM) are carried out to search for magnetic signatures that depend on the carrier density at the interface. Results demonstrate an electronicallycontrolled ferromagnetic phase on top-gated LAO

  16. 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

  17. 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

  18. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    PubMed

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  19. Probing Y-shaped DNA structure with time-resolved FRET

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhasish; Lee, Jong Bum; Valappil, Nikesh V.; Luo, Dan; Menon, Vinod M.

    2012-02-01

    Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between the arms of the Y-DNA and highlighted the overall structural integrity of the Y-DNA system as a leading building block for molecular self-assembly. Temperature dependent lifetime measurements indicated configurational changes in the overall Y-DNA nanoarchitecture above 40 °C.Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between

  20. 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

  1. Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole.

    PubMed

    Tlili, Chaker; Korri-Youssoufi, Hafsa; Ponsonnet, Laurence; Martelet, Claude; Jaffrezic-Renault, Nicole J

    2005-11-15

    We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups. The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone. The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R(2) shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.

  2. Computational and analytical modeling of cationic lipid-DNA complexes.

    PubMed

    Farago, Oded; Grønbech-Jensen, Niels

    2007-05-01

    We present a theoretical study of the physical properties of cationic lipid-DNA (CL-DNA) complexes--a promising synthetically based nonviral carrier of DNA for gene therapy. The study is based on a coarse-grained molecular model, which is used in Monte Carlo simulations of mesoscopically large systems over timescales long enough to address experimental reality. In the present work, we focus on the statistical-mechanical behavior of lamellar complexes, which in Monte Carlo simulations self-assemble spontaneously from a disordered random initial state. We measure the DNA-interaxial spacing, d(DNA), and the local cationic area charge density, sigma(M), for a wide range of values of the parameter (c) representing the fraction of cationic lipids. For weakly charged complexes (low values of (c)), we find that d(DNA) has a linear dependence on (c)(-1), which is in excellent agreement with x-ray diffraction experimental data. We also observe, in qualitative agreement with previous Poisson-Boltzmann calculations of the system, large fluctuations in the local area charge density with a pronounced minimum of sigma(M) halfway between adjacent DNA molecules. For highly-charged complexes (large (c)), we find moderate charge density fluctuations and observe deviations from linear dependence of d(DNA) on (c)(-1). This last result, together with other findings such as the decrease in the effective stretching modulus of the complex and the increased rate at which pores are formed in the complex membranes, are indicative of the gradual loss of mechanical stability of the complex, which occurs when (c) becomes large. We suggest that this may be the origin of the recently observed enhanced transfection efficiency of lamellar CL-DNA complexes at high charge densities, because the completion of the transfection process requires the disassembly of the complex and the release of the DNA into the cytoplasm. Some of the structural properties of the system are also predicted by a continuum

  3. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    PubMed

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  4. DNA tile based self-assembly: building complex nanoarchitectures.

    PubMed

    Lin, Chenxiang; Liu, Yan; Rinker, Sherri; Yan, Hao

    2006-08-11

    DNA tile based self-assembly provides an attractive route to create nanoarchitectures of programmable patterns. It also offers excellent scaffolds for directed self-assembly of nanometer-scale materials, ranging from nanoparticles to proteins, with potential applications in constructing nanoelectronic/nanophotonic devices and protein/ligand nanoarrays. This Review first summarizes the currently available DNA tile toolboxes and further emphasizes recent developments toward self-assembling DNA nanostructures with increasing complexity. Exciting progress using DNA tiles for directed self-assembly of other nanometer scale components is also discussed.

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

    SciTech Connect

    Kreader, C.A.

    1995-04-01

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

  6. Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

    PubMed

    Papadopoulos, Dimitrios K; Krmpot, Aleksandar J; Nikolić, Stanko N; Krautz, Robert; Terenius, Lars; Tomancak, Pavel; Rigler, Rudolf; Gehring, Walter J; Vukojević, Vladana

    2015-11-01

    Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

  7. Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction.

    PubMed

    González, Luis Miguel; Montero, Estrella; Sciutto, Edda; Harrison, Leslie J S; Parkhouse, R Michael E; Garate, Teresa

    2002-04-01

    The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.

  8. Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes.

    PubMed

    Gutmanas, Aleksandras; Billeter, Martin

    2004-12-01

    Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.

  9. Assembly of Slx4 signaling complexes behind DNA replication forks.

    PubMed

    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-08-13

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

  10. 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

  11. DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes.

    PubMed

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Rezvani, Alireza; Mansouri, Ghobad

    2011-05-01

    The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

  12. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures

    SciTech Connect

    Solomon, M.J.; Varshavsky, A.

    1985-10-01

    Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO/sub 4/. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (..cap alpha..-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. It is shown that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the exposed (nuclease-hypersensitive) region of the SV40 chromosomes. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. Other applications of the HCHO technique are discussed, including the possibility of obtaining base-resolution in vivo nucleosome footprints.

  13. Anionic solid lipid nanoparticles supported on protamine/DNA complexes

    NASA Astrophysics Data System (ADS)

    Ye, Jiesheng; Wang, Aihua; Liu, Chunxi; Chen, Zhijin; Zhang, Na

    2008-07-01

    The objective of this study was to design novel anionic ternary nanoparticles for gene delivery. These ternary nanoparticles were equipped with protamine/DNA binary complexes (150-200 nm) as the support, and the anionic formation was achieved by absorption of anionic solid lipid nanoparticles (<=20 nm) onto the surface of the binary complexes. The small solid lipid nanoparticles (SLNs) were prepared by a modified film dispersion-ultrasonication method, and adsorption of the anionic SLNs onto the binary complexes was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7 ± 10.6 nm) with a 'bumpy' surface, and the surface charge inversion from 19.28 ± 1.14 mV to -17.16 ± 1.92 mV could be considered as evidence of the formation of the ternary nanoparticles. The fluorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efficiency of 96.75 ± 1.13%. Circular dichroism spectra analysis showed that the protamine/DNA complexes had been coated by small SLNs, and that the anionic ternary nanoparticles formed did not disturb the construction of the binary complexes. SYBR Green I analysis suggested that the ternary nanoparticles could protect the DNA from nuclease degradation, and cell viability assay results showed that they exhibit lower cytotoxicity to A549 cells compared with the binary complexes and lipofectamine. The transfection efficiency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, and almost equal to that of lipofectamine/DNA complexes, as revealed by inversion fluorescence microscope observation. These results indicated that the anionic ternary nanoparticles could facilitate gene transfer in cultured cells, and might alleviate the drawbacks of the conventional cationic vector/DNA complexes for gene delivery in vivo.

  14. Spin-labeled psoralen probes for the study of DNA dynamics

    SciTech Connect

    Spielmann, H.P.; Chi, D.Y.; Hunt, N.G.

    1995-11-14

    Six nitroxide spin-labeled psoralen derivatives have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-labeled probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of DNA on the time scales investigated, there are no indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralen-cross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity of the adduct. We suggested that the signal for recognition of a lesion to be repaired is in the sugar-phosphate backbone and not in the damaged base(s). 71 refs., 11 figs., 1 tab.

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

    PubMed

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Fang, Xiaohong; Liu, Xiaojing; Tan, Weihong

    1999-05-01

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

  17. Nanopore-based DNA-probe sequence-evolution method unveiling characteristics of protein-DNA binding phenomena in a nanoscale confined space.

    PubMed

    Liu, Nannan; Yang, Zekun; Lou, Xiaoding; Wei, Benmei; Zhang, Juntao; Gao, Pengcheng; Hou, Ruizuo; Xia, Fan

    2015-04-07

    Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein-DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequence-evolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein-DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein-DNA binding and DNA-DNA hybridization.

  18. Fluorogenic boronate-based probe-lactulose complex for full-aqueous analysis of peroxynitrite.

    PubMed

    Li, Kai-Bin; Dong, Lei; Zhang, Siqi; Shi, Wei; Jia, Wen-Ping; Han, De-Man

    2017-04-01

    A selective fluorogenic boronate-based probe-lactulose complex was evaluated for the rapid analysis of peroxynitrite (ONOO(-)) based on a reaction-based indicator displacement assay (RIA). The probe was synthesised by a simple nucleophilic substitution reaction between a boronic acid moiety and a well known laser dye, DCM. Fluorescence analyses showed that the probe had an off-on response to lactulose, forming a fluorogenic probe-lactulose complex. The subsequent addition of ONOO(-) selectively quenched the fluorescence of the complex over other Reactive Oxygen/ Nitrogen Species (ROS/RNS) tested. The complex can be applied for the rapid determination of ONOO(-) in full aqueous solution with good linear range, and has also proven suitable for monitoring ONOO(-) in living cells and real water samples.

  19. Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor.

    PubMed

    Ilkhani, Hoda; Ganjali, Mohamad Reza; Arvand, Majid; Hejazi, Mohammad Saeid; Azimi, Fateme; Norouzi, Parviz

    2011-12-01

    Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl(3) (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)(3). Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)(3) with ds-DNA. It was revealed that Yb(QS)(3) presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)(3) with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10(-8) to 1.1 × 10(-7)M. The interaction mode between Yb(QS)(3) and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.

  20. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  1. DNA probe culture confirmation assay for identification of thermophilic Campylobacter species.

    PubMed Central

    Tenover, F C; Carlson, L; Barbagallo, S; Nachamkin, I

    1990-01-01

    We studied the ability of a new DNA probe-based assay system to correctly identify isolates of the thermophilic campylobacters Campylobacter jejuni, C. coli, and C. laridis grown in vitro. We examined 424 organisms, including 214 Campylobacter isolates and 210 other aerobic and anaerobic isolates. The probe assay, which uses a new homogeneous system in which all reactions take place within a single tube, demonstrated 100% accuracy, producing neither false-positive nor false-negative results. The assay does not, however, distinguish among C. jejuni, C. coli, and C. laridis. PMID:2380357

  2. Cooperative dynamics of a DNA polymerase replicating complex.

    PubMed

    Moors, Samuel L C; Herdewijn, Piet; Robben, Johan; Ceulemans, Arnout

    2013-12-01

    Engineered DNA polymerases continue to be the workhorses of many applications in biotechnology, medicine and nanotechnology. However, the dynamic interplay between the enzyme and the DNA remains unclear. In this study, we performed an extensive replica exchange with flexible tempering (REFT) molecular dynamics simulation of the ternary replicating complex of the archaeal family B DNA polymerase from the thermophile Thermococcus gorgonarius, right before the chemical step. The convoluted dynamics of the enzyme are reducible to rigid-body motions of six subdomains. Upon binding to the enzyme, the DNA double helix conformation changes from a twisted state to a partially untwisted state. The twisted state displays strong bending motion, whereby the DNA oscillates between a straight and a bent conformation. The dynamics of double-stranded DNA are strongly correlated with rotations of the thumb toward the palm, which suggests an assisting role of the enzyme during DNA translocation. In the complex, the primer-template duplex displays increased preference for the B-DNA conformation at the n-2 and n-3 dinucleotide steps. Interactions at the primer 3' end indicate that Thr541 and Asp540 are the acceptors of the first proton transfer in the chemical step, whereas in the translocation step both residues hold the primer 3' terminus in the vicinity of the priming site, which is crucial for high processivity.

  3. Conformational flexibility facilitates self-assembly of complex DNA nanostructures.

    PubMed

    Zhang, Chuan; Su, Min; He, Yu; Zhao, Xin; Fang, Ping-an; Ribbe, Alexander E; Jiang, Wen; Mao, Chengde

    2008-08-05

    Molecular self-assembly is a promising approach to the preparation of nanostructures. DNA, in particular, shows great potential to be a superb molecular system. Synthetic DNA molecules have been programmed to assemble into a wide range of nanostructures. It is generally believed that rigidities of DNA nanomotifs (tiles) are essential for programmable self-assembly of well defined nanostructures. Recently, we have shown that adequate conformational flexibility could be exploited for assembling 3D objects, including tetrahedra, dodecahedra, and buckyballs, out of DNA three-point star motifs. In the current study, we have integrated tensegrity principle into this concept to assemble well defined, complex nanostructures in both 2D and 3D. A symmetric five-point-star motif (tile) has been designed to assemble into icosahedra or large nanocages depending on the concentration and flexibility of the DNA tiles. In both cases, the DNA tiles exhibit significant flexibilities and undergo substantial conformational changes, either symmetrically bending out of the plane or asymmetrically bending in the plane. In contrast to the complicated natures of the assembled structures, the approach presented here is simple and only requires three different component DNA strands. These results demonstrate that conformational flexibility could be explored to generate complex DNA nanostructures. The basic concept might be further extended to other biomacromolecular systems, such as RNA and proteins.

  4. Probing quantum coherence in a biological system by means of DNA amplification.

    PubMed

    Bieberich, E

    2000-07-01

    tunneling as the result of overlapping orbitals along the axis of the primer/template duplex. This effect was unique to the fractal primer due to the number of binding states that remained almost constant, irrespective of the size of shifting. It is suggested that fractal structures found in proteins or other macromolecules may facilitate a short-lived quantum coherent superposition of binding states. This may stabilize molecular complexes for rapid sorting of correct-from-false binding, e.g. during folding or association of macromolecules. The experimental model described in this paper provides a low-cost tool for simulating and probing quantum coherence in a biological system.

  5. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    PubMed

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  6. Visualization of recombinant DNA and protein complexes using atomic force microscopy.

    PubMed

    Murphy, Patrick J M; Shannon, Morgan; Goertz, John

    2011-07-18

    Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation, such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase and a repair enzyme, bound to individual DNA strands. These experiments provide

  7. Optimization of hydrogen bonds for combined DNA/collagen complex.

    PubMed

    Pidaparti, Ramana M; Svintradze, David V; Shan, Yingfeng; Yokota, Hiroki

    2009-01-21

    Many natural and biological systems including collagen and DNA polymers are formed by a process of molecular self-assembly. In this paper, we developed two novel structural models and built heterogeneous DNA/collagen complexes through a preferable arrangement of multiple hydrogen bonds (H-bonds) between DNA and collagen molecules. The simulation results based on three sets of criteria indicate that one of the models with five collagen molecules, which are positioned around each strand of DNA molecules emerged to form a suitable polymer complex with the maximum number of H-bonds. Our predictions quantitatively validated and agreed with the molecular structure reported by Mrevlishvili and Svintradze [2005. Int. J. Biol. Macromol. 36, 324-326].

  8. Electrochemical DNA hybridization sensors applied to real and complex biological samples.

    PubMed

    Tosar, J P; Brañas, G; Laíz, J

    2010-12-15

    DNA hybridization biosensors, also known as genosensors, are analytical devices for the detection of specific DNA "target" sequences in solution, upon hybridization of the targets with complementary "probes" immobilized on a solid substrate. Electrochemical genosensors hold great promise to serve as devices suitable for point-of-care diagnostics and multiplexed platforms for fast, simple and inexpensive nucleic acids analysis. Although a lot of progress has been made in the past few years, the performance of genosensors in complex biological samples has been assayed in only a small fraction of published research articles. This review covers such a group of reports, from the year 2000 onwards. Special attention is played in the nature and complexity of the samples and in the way matrix effects were treated and specificity controls were performed.

  9. The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

    NASA Astrophysics Data System (ADS)

    Xi, Dong; Luo, XiaoPing; Lu, QiangHua; Yao, KaiLun; Liu, ZuLi; Ning, Qin

    2008-03-01

    Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.

  10. Recognition of DNA abasic site nanocavity by fluorophore-switched probe: Suitable for all sequence environments

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Hu, Yuehua; Wu, Tao; Zhang, Lihua; Liu, Hua; Zhou, Xiaoshun; Shao, Yong

    2016-01-01

    Removal of a damaged base in DNA produces an abasic site (AP site) nanocavity. If left un-repaired in vivo by the specific enzyme, this nanocavity will result in nucleotide mutation in the following DNA replication. Therefore, selective recognition of AP site nanocavity by small molecules is important for identification of such DNA damage and development of genetic drugs. In this work, we investigate the fluorescence behavior of isoquinoline alkaloids including palmatine (PAL), berberine (BER), epiberberine (EPI), jatrorrhizine (JAT), coptisine (COP), coralyne (COR), worenine (WOR), berberrubine (BEU), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT) upon binding with the AP nanocavity. PAL is screened out as the most efficient fluorophore-switched probe to recognize the AP nanocavity over the fully matched DNA. Its fluorescence enhancement occurs for all of the AP nanocavity sequence environments, which has not been achieved by the previously used probes. The bridged π conjugation effect should partially contribute to the AP nanocavity-specific fluorescence, as opposed to the solvent effect. Due to the strong binding with the AP nanocavity, PAL will find wide applications in the DNA damage recognition and sensor development.

  11. Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

    PubMed

    Urrutia, R; McNiven, M A; Kachar, B

    1993-05-01

    We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

  12. A reliable method for detecting complexed DNA in vitro

    NASA Astrophysics Data System (ADS)

    Holladay, C.; Keeney, M.; Newland, B.; Mathew, A.; Wang, W.; Pandit, A.

    2010-12-01

    Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-l-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique

  13. Molecular hybridization with DNA-probes as a laboratory diagnostic test for influenza viruses.

    PubMed

    Pljusnin, A Z; Rozhkova, S A; Nolandt, O V; Bryantseva, E A; Kuznetsov, O K; Noskov, F S

    1987-01-01

    The possibilities of using DNA-copies of different influenza A virus genes cloned with recombinant bacterial plasmids for the detection of virus-specific RNA by molecular dot-hybridization were analyzed. High specificity of RNA identification has been demonstrated and it has been shown expedient to use DNA-probes with high-conservative virus genes (polymerase, nucleoprotein, or matrix) for the detection of influenza A virus subtypes (H1N1, H2N2, H3N2) and probes with corresponding hemagglutinin genes for the differentiation of the subtypes H3N2 and H1N1. The results of nasopharyngeal specimens testing proved the effectiveness of molecular dot-hybridization in epidemiological studies of influenza outbreaks, especially of mixed etiology.

  14. Probe classification of on-off type DNA microarray images with a nonlinear matching measure

    NASA Astrophysics Data System (ADS)

    Ryu, Munho; Kim, Jong Dae; Min, Byoung Goo; Kim, Jongwon; Kim, Y. Y.

    2006-01-01

    We propose a nonlinear matching measure, called counting measure, as a signal detection measure that is defined as the number of on pixels in the spot area. It is applied to classify probes for an on-off type DNA microarray, where each probe spot is classified as hybridized or not. The counting measure also incorporates the maximum response search method, where the expected signal is obtained by taking the maximum among the measured responses of the various positions and sizes of the spot template. The counting measure was compared to existing signal detection measures such as the normalized covariance and the median for 2390 patient samples tested on the human papillomavirus (HPV) DNA chip. The counting measure performed the best regardless of whether or not the maximum response search method was used. The experimental results showed that the counting measure combined with the positional search was the most preferable.

  15. A lifetime-sensitive fluorescence anisotropy probe for DNA-based bioassays: The case of SYBR Green.

    PubMed

    Chovelon, Benoit; Fiore, Emmanuelle; Faure, Patrice; Peyrin, Eric; Ravelet, Corinne

    2017-04-15

    In standard steady-state fluorescence anisotropy (FA) DNA-based assays, the ligand binding to a given receptor is typically signalled by the rotational correlation time changes of the tracer. Herein, we report a radically different strategy that relies on the peculiar excited state lifetime features of the SYBR Green (SG) dye. This DNA-binding probe exhibits a drastically short lifetime in solution, leading to a high FA signal. Its complexation to oligonucleotides determines a singular and very large depolarization depending on the concerted effects of extreme lifetime enhancement and resonance energy homotransfer. On the basis of ligand-induced changes in the molar fractions of bound and free forms of SG, the approach provides an unprecedented means for the FA monitoring of the ligand binding to short DNA molecules, allowing the elaboration of a variety of intercalator displacement assays and label-free biosensors that involve diverse DNA structures (duplex, hairpin, G-quadruplex and single-stranded), ligand types (ion, small organic molecule and protein) and binding modes (intercalation, minor groove, allosteric switch). These findings open up promising avenues in the design of a new generation of FA assays.

  16. An osmium-DNA interstrand complex: application to facile DNA methylation analysis.

    PubMed

    Tanaka, Kazuo; Tainaka, Kazuki; Umemoto, Tadashi; Nomura, Akiko; Okamoto, Akimitsu

    2007-11-21

    Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

  17. A probe-based mapping strategy for DNA sequencing with mobile primers

    SciTech Connect

    Strausbaugh, L.D.; Berg, C.M.

    1991-01-01

    Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.

  18. A probe-based mapping strategy for DNA sequencing with mobile primers. Progress report

    SciTech Connect

    Strausbaugh, L.D.; Berg, C.M.

    1991-12-31

    Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.

  19. LL37-DNA complexes and auto-immune diseases

    NASA Astrophysics Data System (ADS)

    Jin, Fan; Sanders, Lori K.; Xian, Wujing; Gilliet, Michel; Wong, Gerard C. L.; Department of Immunology, University of Texas, Houston Collaboration

    2011-03-01

    LL37 is an alpha-helical host defense peptide in humans. Recent work has shown that Toll-like receptor-9 (TLR9), an intracellular receptor in plasmacytoid dendritic cells (pDCs) of the immune system that normally responds to pathogen nucleic acids, can be pathologically triggered by self DNA in the form of DNA-LL37 complexes. Synchrotron small-angle x-ray scattering (SAXS) measurements reveal an unanticipated form of self-assembly between DNA and this positively charged macroion. We examine the generality of this with other macroions, and propose a new geometric criterion for immune cell activation.

  20. Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody.

    PubMed

    Gulis, Galina; Silva, Izabel Cristina Rodrigues; Sousa, Herdson Renney; Sousa, Isabel Garcia; Bezerra, Maryani Andressa Gomes; Quilici, Luana Salgado; Maranhao, Andrea Queiroz; Brigido, Marcelo Macedo

    2016-09-01

    Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

  1. Regulation of DNA Metabolism by DNA-Binding Proteins Probed by Single Molecule Spectroscopy

    DTIC Science & Technology

    2006-12-05

    denaturation The Watson - Crick double- helix is the thermodynamically stable configuration of a DNA molecule under physiological conditions (normal salt and...room/body temperature). This stability is effected (a) by Watson - Crick H-bonding, that is essential for the specificity of base pairing, i.e., for...guarantees the high level of fidelity during replication and transcription. (b) The second contribution to DNA helix stability comes from base-stacking

  2. Contrasting enantioselective DNA preference: chiral helical macrocyclic lanthanide complex binding to DNA.

    PubMed

    Zhao, Chuanqi; Ren, Jinsong; Gregoliński, Janusz; Lisowski, Jerzy; Qu, Xiaogang

    2012-09-01

    There is great interest in design and synthesis of small molecules which selectively target specific genes to inhibit biological functions in which particular DNA structures participate. Among these studies, chiral recognition has been received much attention because more evidences have shown that conversions of the chirality and diverse conformations of DNA are involved in a series of important life events. Here, we report that a pair of chiral helical macrocyclic lanthanide (III) complexes, (M)-Yb[L(SSSSSS)](3+) and (P)-Yb[L(RRRRRR)](3+), can enantioselectively bind to B-form DNA and show remarkably contrasting effects on GC-rich and AT-rich DNA. Neither of them can influence non-B-form DNA, nor quadruplex DNA stability. Our results clearly show that P-enantiomer stabilizes both poly(dG-dC)(2) and poly(dA-dT)(2) while M-enantiomer stabilizes poly(dA-dT)(2), however, destabilizes poly(dG-dC)(2). To our knowledge, this is the best example of chiral metal compounds with such contrasting preference on GC- and AT-DNA. Ligand selectively stabilizing or destabilizing DNA can interfere with protein-DNA interactions and potentially affect many crucial biological processes, such as DNA replication, transcription and repair. As such, bearing these unique capabilities, the chiral compounds reported here may shed light on the design of novel enantiomers targeting specific DNA with both sequence and conformation preference.

  3. The phase behavior of cationic lipid-DNA complexes.

    PubMed Central

    May, S; Harries, D; Ben-Shaul, A

    2000-01-01

    We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

  4. Antitumor drug nogalamycin binds DNA in both grooves simultaneously: molecular structure of nogalamycin-DNA complex.

    PubMed

    Liaw, Y C; Gao, Y G; Robinson, H; van der Marel, G A; van Boom, J H; Wang, A H

    1989-12-26

    The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, d[CGT(pS)ACG] and d[m5CGT(pS)Am5CG], were determined at high resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.

  5. An AFLP-based procedure for the efficient mapping of mutations and DNA probes in barley.

    PubMed Central

    Castiglioni, P; Pozzi, C; Heun, M; Terzi, V; Müller, K J; Rohde, W; Salamini, F

    1998-01-01

    A strategy based upon AFLP markers for high-efficiency mapping of morphological mutations and DNA probes to linkage groups in barley is presented. First, 511 AFLP markers were placed on the linkage map derived from the cross Proctor x Nudinka. Second, loci controlling phenotypic traits were assigned to linkage groups by AFLP analysis, using F2 populations consisting of 30-50 mutant plants derived from crosses of the type "mutant x Proctor" and "mutant x Nudinka." To map DNA probes, 67 different wild-type barley lines were selected to generate F2 populations by crossing with Proctor and Nudinka. F2 plants that were polymorphic for a given RFLP fragment were classified into genotypic classes. Linkage of the RFLP polymorphism to 1 of the 511 AFLP loci was indicated by cosegregation. The use of the strategy is exemplified by the mapping of the mutation branched-5 to chromosome 2 and of the DNA probes Bkn2 and BM-7 to chromosomes 5 and 1, respectively. Map expansion and marker order in map regions with dense clustering of markers represented a particular problem. A discussion considering the effect of noncanonical recombinant products on these two parameters is provided. PMID:9691056

  6. Lanthanide complexes as luminogenic probes to measure sulfide levels in industrial samples.

    PubMed

    Thorson, Megan K; Ung, Phuc; Leaver, Franklin M; Corbin, Teresa S; Tuck, Kellie L; Graham, Bim; Barrios, Amy M

    2015-10-08

    A series of lanthanide-based, azide-appended complexes were investigated as hydrogen sulfide-sensitive probes. Europium complex 1 and Tb complex 3 both displayed a sulfide-dependent increase in luminescence, while Tb complex 2 displayed a decrease in luminescence upon exposure to NaHS. The utility of the complexes for monitoring sulfide levels in industrial oil and water samples was investigated. Complex 3 provided a sensitive measure of sulfide levels in petrochemical water samples (detection limit ∼ 250 nM), while complex 1 was capable of monitoring μM levels of sulfide in partially refined crude oil.

  7. Visualization of DNA and Protein-DNA Complexes with Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Gall, Alexander A.; Shlyakhtenko, Luda S.

    2014-01-01

    This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes. PMID:24357372

  8. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  9. BaitFisher: A Software Package for Multispecies Target DNA Enrichment Probe Design.

    PubMed

    Mayer, Christoph; Sann, Manuela; Donath, Alexander; Meixner, Martin; Podsiadlowski, Lars; Peters, Ralph S; Petersen, Malte; Meusemann, Karen; Liere, Karsten; Wägele, Johann-Wolfgang; Misof, Bernhard; Bleidorn, Christoph; Ohl, Michael; Niehuis, Oliver

    2016-07-01

    Target DNA enrichment combined with high-throughput sequencing technologies is a powerful approach to probing a large number of loci in genomes of interest. However, software algorithms that explicitly consider nucleotide sequence information of target loci in multiple reference species for optimizing design of target enrichment baits to be applicable across a wide range of species have not been developed. Here we present an algorithm that infers target DNA enrichment baits from multiple nucleotide sequence alignments. By applying clustering methods and the combinatorial 1-center sequence optimization to bait design, we are able to minimize the total number of baits required to efficiently probe target loci in multiple species. Consequently, more loci can be probed across species with a given number of baits. Using transcript sequences of 24 apoid wasps (Hymenoptera: Crabronidae, Sphecidae) from the 1KITE project and the gene models of Nasonia vitripennis, we inferred 57,650, 120-bp-long baits for capturing 378 coding sequence sections of 282 genes in apoid wasps. Illumina reduced-representation library sequencing confirmed successful enrichment of the target DNA when applying these baits to DNA of various apoid wasps. The designed baits furthermore enriched a major fraction of the target DNA in distantly related Hymenoptera, such as Formicidae and Chalcidoidea, highlighting the baits' broad taxonomic applicability. The availability of baits with broad taxonomic applicability is of major interest in numerous disciplines, ranging from phylogenetics to biodiversity monitoring. We implemented our new approach in a software package, called BaitFisher, which is open source and freely available at https://github.com/cmayer/BaitFisher-package.git.

  10. Accuracy of the Clinical Diagnosis of Vaginitis Compared to a DNA Probe Laboratory Standard

    PubMed Central

    Lowe, Nancy K.; Neal, Jeremy L.; Ryan-Wenger, Nancy A.

    2009-01-01

    Objective To estimate the accuracy of the clinical diagnosis of the three most common causes of acute vulvovaginal symptoms (bacterial vaginosis, candidiasis vaginitis, and trichomoniasis vaginalis) using a traditional, standardized clinical diagnostic protocol compared to a DNA probe laboratory standard. Methods This prospective clinical comparative study had a sample of 535 active duty United States military women presenting with vulovaginal symptoms. Clinical diagnoses were made by research staff using a standardized protocol of history, physical examination including pelvic examination, determination of vaginal pH, vaginal fluid amines test, and wet-prep microscopy. Vaginal fluid samples were obtained for DNA analysis. The research clinicians were blinded to the DNA results. Results The participants described a presenting symptom of abnormal discharge (50%), itching/irritation (33%), malodor (10%), burning (4%), or others such as vulvar pain and vaginal discomfort. According to laboratory standard, there were 225 cases (42%) of bacterial vaginosis 76 cases (14%) of candidiasis vaginitis, 8 cases (1.5%) of trichomoniasis vaginalis, 87 cases of mixed infections (16%), and 139 negative cases (26%). For each single infection, the clinical diagnosis had a sensitivity and specificity of 80.8% and 70.0% for bacterial vaginosis; 83.8% and 84.8% for candidiasis vaginitis; and 84.6% and 99.6% for trichomoniasis vaginalis when compared to the DNA probe standard. Conclusion Compared to a DNA probe standard, clinical diagnosis is 81-85% sensitive and 70- 99% specific for bacterial vaginosis, candida vaginitis, and trichomoniasis. Even under research conditions that provided clinicians with sufficient time and materials to conduct a thorough and standardized clinical evaluation, the diagnosis and therefore, subsequent treatment of these common vaginal problems remains difficult. PMID:19104364

  11. Dynamics of the binding of acridine dyes to DNA investigated by triplet excited state probe techniques

    SciTech Connect

    Geacintov, N.E.; Waldmeyer, J.; Kuzmin, V.A.; Kolubayev, T.

    1981-11-26

    The binding of the polynuclear aromatic dyes acridine orange (AO) and proflavin (PF) to DNA in aqueous phosphate buffer solution at 25 +- 1/sup 0/C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes can be measured either by triplet-triplet absorption flash photolysis techniques or by delayed fluorescence methods. The triplet lifetimes of AO vary from about 0.5 ms with no DNA present to 20 to 35 ms at DNA concentration above 10/sup -3/M expressed in concentration of DNA phosphate (P), or at (P)/(D) ratios above 1000 ((D) is the dye concentration). At all DNA concentrations the decay profiles are exponential, except at high excitation intensities where nonexponentialities, attributed to triplet-triplet annihilation, become apparent. Similar results are observed with PF-DNA solutions. The exponentiality of the triplet decay at all DNA concentrations is attributed to rapid association and dissociation of the dye-DNA complexes on the time scales of the triplet lifetimes. A simplified one-step binding model is utilized to describe this effect. A dissociation rate of AO-DNA complexes greater than or equal to 10/sup 3/s/sup -1/ has been estimated from these results. It is shown that a detailed study of the triplet lifetime vs. DNA concentration provides a novel method for the estimation of the apparent equilibrium association constant K* for dye molecules in the triplet excited state and DNA. For AO, K* approx. = 10/sup 5/ M/sup -1/, while for PF it is approx. = 3 x 10/sup 4/ M/sup -1/. These values are of the same order of magnitude as the ground-state dye-DNA equilibrium association constants measured by others.

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

    NASA Astrophysics Data System (ADS)

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

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

  13. Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

    PubMed

    Love, D N; Bailey, G D; Bastin, D

    1992-06-01

    A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

  14. Complexation of heterocyclic ligands with DNA in aqueous solution

    NASA Astrophysics Data System (ADS)

    Baranovskii, S. F.; Bolotin, P. A.; Evstigneev, M. P.; Chernyshev, D. N.

    2008-03-01

    We have used spectrophotometry to study self-association and complexation with DNA by organic heterocyclic compounds in the acridine and phenothiazine series: proflavin, thionine, and methylene blue. Based on the experimental concentration dependences of the molar absorption coefficient of the molecules in an aqueous buffer solution (0.01 M NaCl, 0.01 M Na2EDTA, 0.01 M Tris, pH 7.4, T = 298 K), we have determined the equilibrium dimerization constants for the dyes and the DNA complexation parameters using the Scatchard and McGhee-von Hippel models. The observed increase in the cooperativity parameters as the dimerization constants of the ligands increase allowed us to hypothesize that the same interactions occur between dye molecules adsorbed on DNA as in their self-association. The equilibrium DNA-binding constants for the ligands, obtained using the McGhee-von Hippel cooperative model, are (20.9 ± 2.7)·103 M-1 for proflavin and (33.8 ± 4.1)·103 M-1 for thionine. Using the Scatchard model, taking into account intercalation and “external” binding of ligands with DNA, we determined the DNA complexation constants for methylene blue: (26.4 ± 4.6)·103 and (96 ± 17)·103 M-1 respectively. Based on analysis of the data obtained, we hypothesized that the predominant type of binding with DNA is intercalation binding in the case of proflavin and thionine, and “external” binding with the DNA surface in the case of methylene blue.

  15. Viral Contribution to Dissolved DNA in the Marine Environment as Determined by Differential Centrifugation and Kingdom Probing

    PubMed Central

    Jiang, S. C.; Paul, J. H.

    1995-01-01

    Dissolved or filterable (<0.2-(mu)m-pore-size filter) DNA is a ubiquitous component of the dissolved organic matter in the surface waters of this planet. In an effort to understand the composition and possible sources, we subjected dissolved DNA concentrated by vortex flow filtration from offshore and coastal environments to differential centrifugation and probing with 16S rRNA-targeted kingdom oligonucleotide probes. Initial studies with calf thymus soluble DNA and T2 phage particles indicated that high-speed ultracentrifugation (201,000 x g for 90 min), a method to separate viral particles from soluble DNA used by other investigators, resulted in pelleting of nearly all the DNA and virus particles. Lower-speed centrifugation (11,200 to 25,800 x g for 90 min) resulted in >99% of the virus particles being collected in the pellet and (equiv)65% of the calf thymus DNA remaining in the supernatant. Employing this approach, we estimate that approximately 50% of the filterable DNA from marine environments is truly soluble or free DNA and that the other half is composed of bound forms (viral particles and, potentially, colloids). Of the bound form, 17 to 30% could be accounted for by viral particles, by calculating the amount of viral DNA on the basis of viral abundance, leaving a portion of the bound form uncharacterized. Kingdom probing with universal, eubacterial, and eucaryotic probes indicated that dissolved DNA hybridized with all of these probes, while purified standard viral DNAs did not, or hybridized only slightly with the universal probe (tailed oligonucleotide only). Collectively, these data indicate that DNA in viral particles is a small component of the dissolved DNA, the majority being of eubacterial and eucaryotic origin. PMID:16534913

  16. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  17. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2012-03-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  18. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2011-11-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  19. VCD spectroscopy as an excellent probe of chiral metal complexes containing a carbon monoxide vibrational chromophore.

    PubMed

    Fusè, Marco; Mazzeo, Giuseppe; Longhi, Giovanna; Abbate, Sergio; Zerla, Daniele; Rimoldi, Isabella; Contini, Alessandro; Cesarotti, Edoardo

    2015-06-07

    Vibrational circular dichroism, VCD, gives evidence that the carbon monoxide chromophore in a heteroleptic cyclopentadienyl Ru(ii)-carbonyl complex is very sensitive to the chirality of the metal centre and becomes an excellent probe to define the configuration of chiral metal complexes.

  20. Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

    PubMed Central

    Wang, Xiaoqing; Sharp, Christine E.; Jones, Gareth M.; Grasby, Stephen E.; Brady, Allyson L.

    2015-01-01

    The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. 13C-labeled EPS and 13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from 13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species. PMID:25934620

  1. A novel DNA tetrahedron-hairpin probe for in situ"off-on" fluorescence imaging of intracellular telomerase activity.

    PubMed

    Feng, Qiu-Mei; Zhu, Meng-Jiao; Zhang, Ting-Ting; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-21

    A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.

  2. TOPOLOGIES OF COMPLEXES CONTAINING O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE AND DNA

    PubMed Central

    Adams, Claire A.; Melikishvili, Manana; Rodgers, David W.; Rasimas, Joseph J.; Pegg, Anthony E.; Fried, Michael G.

    2009-01-01

    SUMMARY The mutagenic and cytotoxic effects of many alkylating agents are reduced by O6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multi-protein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a 3-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein crosslinking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions. PMID:19358853

  3. Fluorescence Probe for Detecting CCG Trinucleotide Repeat DNA Expansion and Slip-Out.

    PubMed

    Shibata, Tomonori; Nakatani, Kazuhiko

    2016-09-15

    Trinucleotide repeat expansion in genomic DNA causes severe neurodegenerative diseases. Fluorescence probes that bind to trinucleotide repeats have potential as diagnostic tools of trinucleotide repeat disorders. Here, we report a novel tricyclic ligand that binds to CCG trinucleotide repeat DNA. The expansion of the aromatic ring system of the 2-amino-1,8-naphthyridine chromophore from the bicyclic to the tricyclic improved the binding ability to the CCG/CCG motif without losing the selectivity and emissive character. The fluorescence sensitively decreased in response to binding to the CCG trinucleotide repeat. The degree of quenching depended on the number of CCG repeats. In addition, the fluorescence detection was applicable to CCG slip-out DNA.

  4. Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification.

    PubMed

    Ehlert, Alexandra; Demmel, Anja; Hupfer, Christine; Busch, Ulrich; Engel, Karl-Heinz

    2009-04-01

    The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg(-1) range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg(-1). The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

  5. Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats.

    PubMed

    Love, D N; Bailey, G D

    1993-01-01

    A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific--differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

  6. Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

    NASA Astrophysics Data System (ADS)

    Monico, Carina; Capitanio, Marco; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.

    2013-06-01

    We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the kinetics of both short- and long-lived interactions of LacI along the DNA template (from ˜100 μs to tens of seconds), as well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other single-molecule methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.

  7. Photocatalytic probing of DNA sequence by using TiO{sub 2}/dopamine-DNA triads.

    SciTech Connect

    Liu, J.; de la Garza, L.; Zhang, L.; Dimitrijevic, N. M.; Zuo, X.; Tiede, D. M.; Rajh, T.

    2007-10-15

    A method to control charge transfer reaction in DNA using hybrid nanometer-sized TiO{sub 2} nanoparticles was developed. In this system extended charge separation reflects the sequence of DNA and was measured using metallic silver deposition or by photocurrent response. Light-induced extended charge separation in these systems was found to be dependent on the DNA-bridge length and sequence. The yield of photocatalytic deposition of silver was studied in systems having GG accepting sites imbedded in AT runs at varying distances from the TiO{sub 2} nanoparticle surface. Weak distance dependence of charge separation indicative of a hole hopping through mediating adenine (A) sites was found. The quantum yield of silver deposition in the system having a GG accepting site placed 8.5 {angstrom} from the nanoparticle surface was found to be {Phi} = 0.70 (70%) and {Phi} = 0.56 (56%) for (A){sub n} and (AT){sub n/2} bridge, respectively. Hole injection to GG trapping sites as far as 70 {angstrom} from a nanoparticle surface in the absence of G hopping sites was measured. Introduction of G hopping sites increased the efficiency of hole injection. The efficiency of photocatalytic deposition of metallic silver was found to be sensitive to the presence of a single nucleobase mismatch in the DNA sequence.

  8. Linear Dichroism Characteristics of Ethidium and Proflavine Supercoiled DNA Complexes

    DTIC Science & Technology

    1990-01-01

    of Ethidium-and Proflavine -Supercoi led DNA Complexes CHARLES E. SWENBERG, 1 SUSAN E. CARBERRV, 2 * and NICHOLAS E. GEACINTOV’ SRadliationl Biochenitr...drug were stained with EB, photographed under uv light, molecules, EB and proflavine (PF) (Figure 1), and the bands were quantitated by densitometry

  9. A platinum complex that binds non-covalently to DNA and induces cell death via a different mechanism than cisplatin.

    PubMed

    Suntharalingam, Kogularamanan; Mendoza, Oscar; Duarte, Alexandra A; Mann, David J; Vilar, Ramon

    2013-05-01

    Cisplatin and some of its derivatives have been shown to be very successful anticancer agents. Their main mode of action has been proposed to be via covalent binding to DNA. However, one of the limitations of these drugs is their poor activity against some tumours due to intrinsic or acquired resistance. Therefore, there is interest in developing complexes with different binding modes and mode of action. Herein we present a novel platinum(ii)-terpyridine complex (1) which interacts non-covalently with DNA and induces cell death via a different mechanism than cisplatin. The interaction of this complex with DNA was studied by UV/Vis spectroscopic titrations, fluorescent indicator displacement (FID) assays and circular dichroism (CD) titrations. In addition, computational docking studies were carried out with the aim of establishing the complex's binding mode. These experimental and computational studies showed the complex to have an affinity constant for DNA of ∼10(4) M(-1), a theoretical free energy of binding of -10.83 kcal mol(-1) and selectivity for the minor groove of DNA. Long-term studies indicated that 1 did not covalently bind (or nick) DNA. The cancer cell antiproliferative properties of this platinum(ii) complex were probed in vitro against human and murine cell lines. Encouragingly the platinum(ii) complex displayed selective toxicity for the cancerous (U2OS and SH-SY5Y) and proliferating NIH 3T3 cell lines. Further cell based studies were carried out to establish the mode of action. Cellular uptake studies demonstrated that the complex is able to penetrate the cell membrane and localize to the nucleus, implying that genomic DNA could be a cellular target. Detailed immunoblotting studies in combination with DNA-flow cytometry showed that the platinum(ii) complex induced cell death in a manner consistent with necrosis.

  10. Intercalation between antitumor anthracyclines and DNA as probed by resonance and surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Smulevich, G.; Mantini, A. R.; Casu, M.; Marzocchi, M. P.

    1991-05-01

    The antiturnor anthracyclincs, idarubicin (IDA ), adrianiycin (ADM), epirubicin (EPI), carminomycin (CAR) and 1 1-deoxycarminornycin (DCM), whose siructural formula includes a substituted hydroxyanthraquirionc chrornophore and a sugar residue, form intercalation complexes with DNA. The stacking interaction between the chromophore and the base-pairs of DNA gives rise to noticeable ciTects on resonance Raman (RR) and surface-enhanced resonance Raman (SERRS) scattering as well as on the absorption (ABS), its second derivative (D2) and fluorescence emission (FEM) spectra.

  11. Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.

    PubMed

    Rojowska, Anna; Lammens, Katja; Seifert, Florian U; Direnberger, Carolin; Feldmann, Heidi; Hopfner, Karl-Peter

    2014-12-01

    The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.

  12. High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

    NASA Astrophysics Data System (ADS)

    Weier, Heinz-Ulli G.; Munne, S.; Lersch, Robert A.; Marquez, C.; Wu, J.; Pedersen, Roger A.; Fung, Jingly

    1999-06-01

    The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in

  13. Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

    NASA Astrophysics Data System (ADS)

    Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2017-03-01

    Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

  14. M13 repeat probe detects DNA minisatellite-like sequences in gymnosperms and angiosperms.

    PubMed

    Rogstad, S H; Patton, J C; Schaal, B A

    1988-12-01

    Several kinds of minisatellite DNA, all of which are composed of low to moderately repetitive DNA, have been identified in tetrapod genomes. While the repeating oligonucleotide elements (subrepeats) of a given minisatellite are virtually identical, subrepeat nucleotide composition differs between different minisatellites. Several minisatellites have exhibited moderate to high levels of restriction length polymorphism in a number of tetrapods. Such hypervariable markers provide powerful tools for genetic analyses in several fields of biology. Minisatellite applications have been restricted to tetrapods, but here we demonstrate that one probe, the M13 repeat probe previously used to detect minisatellites in humans and bovines, also reveals minisatellite-bearing endonuclease fragments in gymnosperms and angiosperms. While the plant minisatellites appear to be somatically stable within an individual, they often vary within species in potentially useful ways. These results demonstrate that minisatellite-like families may be distributed over a wide taxonomic range in eukaryotes, opening the possibility of a commensurately wide utility of minisatellite probes in genetic analyses.

  15. Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes.

    PubMed

    Lu, Lihua; Zhong, Hai-Jing; Wang, Modi; Ho, See-Lok; Li, Hung-Wing; Leung, Chung-Hang; Ma, Dik-Lung

    2015-09-30

    We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aβ1-40 peptide aggregation in vitro, and protected against Aβ-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aβ1-40 peptide on the basis of their emission response.

  16. Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes

    PubMed Central

    Lu, Lihua; Zhong, Hai-Jing; Wang, Modi; Ho, See-Lok; Li, Hung-Wing; Leung, Chung-Hang; Ma, Dik-Lung

    2015-01-01

    We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aβ1–40 peptide aggregation in vitro, and protected against Aβ-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aβ1–40 peptide on the basis of their emission response. PMID:26419607

  17. Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes

    NASA Astrophysics Data System (ADS)

    Lu, Lihua; Zhong, Hai-Jing; Wang, Modi; Ho, See-Lok; Li, Hung-Wing; Leung, Chung-Hang; Ma, Dik-Lung

    2015-09-01

    We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aβ1-40 peptide aggregation in vitro, and protected against Aβ-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aβ1-40 peptide on the basis of their emission response.

  18. Probing nuclear pore complex architecture with proximity-dependent biotinylation.

    PubMed

    Kim, Dae In; Birendra, K C; Zhu, Wenhong; Motamedchaboki, Khatereh; Doye, Valérie; Roux, Kyle J

    2014-06-17

    Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.

  19. Using Electron Distributions to Probe Energy Surfaces at Complex R

    NASA Astrophysics Data System (ADS)

    Macek, J. H.; Ovchinnikov, S. Yu.

    1997-04-01

    The hidden crossing theory describes ion-atom collisions(S. Yu. Ovchinnikov and E. A. Solovév, Comments At. Mol. Phys. 22) 69 (1988). in terms of a single function \\varepsilon(R) defined for all complex R, where R is the distance between target and projectile nuclei. Conventional adiabatic energy curves \\varepsilon_n(R) represent different branches of \\varepsilon(R) at real, positive R. Electron distributions are computed by evaluating a phase integral along an appropriate path in the complex R-plane. The real part of the phase oscillates rapidly for a class of transitions that proceed via the "top of barrier" mechanism. Electron distributions oscillate owing to interfrence between σ and π transitions, and this oscillation relates closely to the real part of \\varepsilon(R) for complex R. The oscillation rate is in qualitative agreement with measurements (R. Döner, K. Khemliche, M. H. Prior, C. L. Cocke, J. A. Gary, R. E. Olson, V. Mergel, J. Ullrich and H. Schmidt-Böking, Phys. Rev. Lett.77), 1024 (1996).

  20. Probing Potential Energy Surface Exploration Strategies for Complex Systems.

    PubMed

    N'Tsouaglo, Gawonou Kokou; Béland, Laurent Karim; Joly, Jean-François; Brommer, Peter; Mousseau, Normand; Pochet, Pascal

    2015-04-14

    The efficiency of minimum-energy configuration searching algorithms is closely linked to the energy landscape structure of complex systems, yet these algorithms often include a number of steps of which the effect is not always clear. Decoupling these steps and their impacts can allow us to better understand both their role and the nature of complex energy landscape. Here, we consider a family of minimum-energy algorithms based, directly or indirectly, on the well-known Bell-Evans-Polanyi (BEP) principle. Comparing trajectories generated with BEP-based algorithms to kinetically correct off-lattice kinetic Monte Carlo schemes allow us to confirm that the BEP principle does not hold for complex systems since forward and reverse energy barriers are completely uncorrelated. As would be expected, following the lowest available energy barrier leads to rapid trapping. This is why BEP-based methods require also a direct handling of visited basins or barriers. Comparing the efficiency of these methods with a thermodynamical handling of low-energy barriers, we show that most of the efficiency of the BEP-like methods lie first and foremost in the basin management rather than in the BEP-like step.

  1. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    SciTech Connect

    Walia, S.; Khan, A.; Rosenthal, N. )

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.

  2. Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response.

    PubMed

    Crnolatac, Ivo; Rogan, Iva; Majić, Boris; Tomić, Sanja; Deligeorgiev, Todor; Horvat, Gordan; Makuc, Damjan; Plavec, Janez; Pescitelli, Gennaro; Piantanida, Ivo

    2016-10-12

    Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400-500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU.

  3. Probing Complexity using the LCLS and the ALS

    SciTech Connect

    Berrah, Nora

    2015-02-19

    The goal of our research program is to investigate fundamental interactions between photons and molecular/nano-systems to advance our quantitative understanding of electron correlations, charge transfer and many body phenomena. Our research projects focus on probing, on a femtosecond time-scale, multi-electron interactions and tracing nuclear motion in order to understand, and ultimately control energy flow and charge transfer processes from electromagnetic radiation to matter. The experiments will be carried out with state of the art instrumentation built by the P.I. team with funds from a DoE "Single Investigator and Small Group Research" (SISGR) grant. The research projects carried out the past three years consisted of first experiments using the linac coherent light source (LCLS) x-ray free electron laser (FEL) facility at the SLAC National Laboratory, as well as the study of correlated processes in select anions using the ALS. A report for the past cycle is described in section II. These studies have paved the way for our renewal application for the next three years. Our research interests for the next three years extend our past and present research by carrying out time-resolved measurements described in section III. They will consist of: a) The study of molecular dynamics that happen on ultrafast time scales, using pump-probe schemes and the study of non-linear physics in the x-ray regime via multi-photon absorption from the LCLS. This will be achieved by measuring and examining both electronic and nuclear dynamics subsequent to the interaction of molecules and nano-systems with LCLS pulses of various wavelength, intensity and pulse duration as described in section III.A. b) The study of molecular dynamics and correlated processes via absorption of vuv-soft x-rays from the Advanced Light Source (ALS) at Lawrence Berkeley Laboratory to provide single-photon ionization baseline results for LCLS studies. In addition, we will study the photodetachment of anions

  4. Meat speciation by restriction fragment length polymorphism analysis using an α-actin cDNA probe.

    PubMed

    Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

    1998-09-01

    Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic 'fingerprints' which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

  5. A electrogenerated chemiluminescence biosensor for Ramos cancer cell using DNA encapsulated Ru(bpy)₃Cl₂ as signal probe.

    PubMed

    Hun, Xu; Chen, Huaicheng; Wang, Wei

    2011-05-15

    A label-free sensing technology for detection of Ramos cell was developed based on a signal probe Ru(bpy)3Cl2 (Ru) encapsulated by DNA. Gold electrode or magnetic bead as the sensing surface was firstly modified with long-strand DNA with five repeating units. Then two kinds of short-strand DNA are grafted onto the long-strand DNA to form DNA strands A and B (L-A and L-B) through the hybridization, respectively. The addition of aptamer initiates hybridization of L-A and L-B with the aptamer sequence. As the hybridization proceeds, the four kinds of DNA would finally transform into a three-dimensional network structure and the signal probe Ru was encapsulated by DNA simultaneously. When Ramos cells are introduced to interact with the aptamer, the signal probe is released. In order to confirm the generality of this method the ferrocenecarboxylic acid and luminol selected as a signal probe mode were also tested. The Ru used as a signal probe for electrogenerated chemiluminescence (ECL) detection was detailedly studied. With this ECL biosensor, detection limit as low as 58 cells/mL was achieved for Ramos cell. The biosensor also exhibited excellent sensitivity and selectivity.

  6. [Genetic diversity of 3 DNA probes in the DNA fingerprinting of a Mexican population].

    PubMed

    Berumen-Campos, J; Casas-Avila, L; Hernández-Mendoza, A; Segura-Salinas, E; Medina-León, R; Larriva-Sahd, J

    1994-01-01

    Each individual may be identified by characterizing its genetic material by DNA fingerprinting technology. Its application in Mexico demands a knowledge of the allelic and genotypic diversity of the DNA markers and the probability that two individuals may have the same fingerprint. In the present study the allelic and genotypic diversities of the loci D12S11 (MS43A), D7S22 (g3) and D1S7 (MS1) were determined in 100 Mexican students of the military school of medicine (Escuela Médico Militar de México). The mean allelic frequency of the loci MS43A, g3, and MS1 was 0.01, 0.008 and 0.006, respectively. The heterozygosity of MS43A and g3 was 98 and 99% for MS1. The probability that two individuals might have the same genetic pattern was 2.0 x 10(-4), 1.3 x 10(-4) and 7.2 x 10(-5) for the loci MS43A, g3 and MS1, respectively, and as low as 1.9 x 10(-12) for the three taken together. These data indicate that the genetic diversity of these DNA fingerprinting markers in the Mexican population is high enough to warrant its use in paternity testing and in the identification of individuals in forensic medicine.

  7. An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices

    PubMed Central

    Xia, Fan; White, Ryan J.; Zuo, Xiaolei; Patterson, Adriana; Xiao, Yi; Kang, Di; Gong, Xiong; Plaxco, Kevin W.; Heeger, Alan J.

    2011-01-01

    In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a “sticky end.” When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry. PMID:20873767

  8. Protein-free parallel triple-stranded DNA complex formation

    PubMed Central

    Shchyolkina, A. K.; Timofeev, E. N.; Lysov, Yu. P.; Florentiev, V. L.; Jovin, T. M.; Arndt-Jovin, D. J.

    2001-01-01

    A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed. PMID:11160932

  9. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes.

    PubMed

    McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L; Pegg, Ian L; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether's extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule's force response. Using a calibration technique based on Stokes' drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level.

  10. Fidelity of a human cell DNA replication complex.

    PubMed Central

    Roberts, J D; Kunkel, T A

    1988-01-01

    We have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ alpha-complementation gene, which encodes the alpha region (specifying the amino-terminal portion) of beta-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase alpha-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes. Images PMID:3174620

  11. Unwrapping of DNA-protein complexes under external stretching.

    PubMed

    Sakaue, Takahiro; Löwen, Hartmut

    2004-08-01

    A DNA-protein complex modeled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without a stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and dynamics of the unwrapping process by computer simulations and simple phenomenological theory. We find two different scenarios depending on the chain stiffness: For a flexible chain, the extension of the complex scales linearly with the external force applied. The sphere-chain complex is disordered; i.e., there is no clear winding of the chain around the sphere. For a stiff chain, on the other hand, the complex structure is ordered, which is reminiscent of nucleosome. There is a clear winding number, and the unwrapping process under external stretching is discontinuous with jumps of the distance-force curve. This is associated with discrete unwinding processes of the complex. Our predictions are of relevance for experiments, which measure force-extension curves of DNA-protein complexes, such as nucleosome, using optical tweezers.

  12. Ultrafast excited-state dynamics at interfaces: fluorescent DNA probes at the dodecane/water interface

    NASA Astrophysics Data System (ADS)

    Licari, Giuseppe; Vauthey, Eric

    2015-08-01

    Although the interfaces between two isotropic media are of primary importance in many areas of science and technology, their properties are only partially understood. Our strategy to obtain an insight into these properties is to investigate the ultrafast excited-state dynamics of environment-sensitive molecular probes at liquid interfaces using time-resolved surface second harmonic generation, and to compare it with the dynamics of the same molecules in bulk solutions. Additionally, this approach gives rich information on how the chemical reactivity may change when going from the bulk phase to the interface. This is illustrated by an investigation performed with a series of fluorescent DNA probes at the dodecane/water interface without and with the presence of DNA in the aqueous phase. Substantial differences in the conformation of these cyanine dyes (aggregated or not) and in the excited-state dynamics are observed when going from bulk solutions to the interface. Moreover, the presence of double-stranded DNA in the aqueous phase induces some chirality at the interface.

  13. Non-Natural Nucleotides as Probes for the Mechanism and Fidelity of DNA Polymerases

    PubMed Central

    Lee, Irene; Berdis, Anthony J.

    2009-01-01

    DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides. PMID:19733263

  14. DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Krajewska, Barbara; Willemeit, Markus; Deml, Alexander; Klösel, Roland; Rodríguez, Julio R

    2010-07-29

    The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

  15. Relocalization of DNA lesions to the nuclear pore complex

    PubMed Central

    Freudenreich, Catherine H.; Su, Xiaofeng A.

    2016-01-01

    Early screens in yeast for mutations exhibiting sensitivity to DNA damage identified nuclear pore components, but their role in DNA repair was not well understood. Over the last decade, studies have revealed that several types of persistent DNA lesions relocate to either the nuclear pore complex (NPC) or nuclear envelope (NE). Of these two sites, the nuclear pore appears to be crucial for DNA repair of persistent double-strand breaks, eroded telomeres and sites of fork collapse at expanded CAG repeats. Using a combination of cell biological imaging techniques and yeast genetic assays for DNA repair, researchers have begun to understand both the how and why of lesion relocation to the NPC. Here we review the types of lesions that relocate to the NPC, mediators of relocation and the functional consequences of relocation understood to date. The emerging theme is that relocation to the NPC regulates recombination to influence repair pathway choice and provide a rescue mechanism for lesions or DNA structures that are resistant to repair. PMID:27799300

  16. Efficient model of an open-ended coaxial-line probe for measuring complex permittivity

    NASA Astrophysics Data System (ADS)

    Shin, Hyun; Hyun, Seung-Yeup; Kim, Sang-Wook; Kim, Se-Yun

    2000-07-01

    A virtually conical cable model of an open-ended coaxial- line probe for converting its measured reflection coefficients into the complex permittivity of a contacted material is presented here. Both reflection coefficients of air and pure water are calculated by employing the FDTD method, and the phase difference between the calculated and the measured reflection coefficients of pure water is used as a calibration factor of the probe. The virtually conical cable model renders the conversion of the complex permittivity of dry sand more accurate and faster than the integral equation model to the aperture admittance.

  17. DNA-osmium complexes: recent developments in the operative chemical analysis of DNA epigenetic modifications.

    PubMed

    Okamoto, Akimitsu

    2014-09-01

    The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine-osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.

  18. [Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy].

    PubMed

    Klinov, D V; Martynkina, L P; Iurchenko, V Iu; Demin, V V; Strel'tsov, S A; Gerasimov, Iu A; Vengerov, Iu Iu

    2003-01-01

    Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.

  19. Noncovalent attachment of psoralen derivatives with DNA: Hartree-Fock and density functional studies on the probes

    NASA Astrophysics Data System (ADS)

    El-Gogary, Tarek M.; El-Gendy, Eman M.

    2003-09-01

    Two psoralen derivatives (probes) were prepared. Their geometries were optimized at the Hartree-Fock (HF) and Density Functional (B3LYP) levels employing 6-31G** and cc-pVDZ basis sets. Their interaction with DNA was investigated using spectrophotometric and computational techniques. Both of them have shown strong binding to calf thymus DNA. The red-shift and hypochromism that detected in the spectrum were taken as an evidence for the strong interaction between these probes and DNA. The spectrophotometric DNA titration data were treated by two different methodologies to calculate the intercalation affinity. Half-reciprocal plots gave binding constants of 5.5065×10 4 and 6.4727×10 4 for 8-butoxypsoralen (8-BOP) and 8-hexoxypsoralen (8-HOP), respectively. Schatchard plots gave a comparable intercalation binding constants and also the surface binding constants along with the number of intercalated probe molecules per base pair. The interaction between these probes and DNA were studied theoretically. The energy of interaction was computed using molecular mechanics method. Strength of interaction of these probes with different types of DNA was computed and compared. Calculated energies of interaction were compared with the observed intercalation affinities. HOMO and LUMO energies were computed and used to account for the strength of interaction.

  20. New insights into the transition pathway from nonspecific to specific complex of DNA with Escherichia coli integration host factor.

    PubMed

    Vivas, Paula; Kuznetsov, Serguei V; Ansari, Anjum

    2008-05-15

    To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate

  1. Complex Archimedean tiling self-assembled from DNA nanostructures.

    PubMed

    Zhang, Fei; Liu, Yan; Yan, Hao

    2013-05-22

    Archimedean tilings are periodic polygonal tessellations that are created by placing regular polygons edge-to-edge around a vertex to fill the plane. Here we show that three- and four-arm DNA junction tiles with specifically designed arm lengths and intertile sticky-end interactions can be used to form sophisticated two-dimensional (2D) and three-dimensional (3D) tessellation patterns. We demonstrate two different complex Archimedean patterns, (3(3).4(2)) and (3(2).4.3.4), and the formation of 2D lattices, 3D tubes, and sealed polygon-shaped pockets from the tessellations. The successful growth of hybrid DNA tile motif arrays suggests that it maybe possible to generate 2D quasi-crystals from DNA building blocks.

  2. Identification of Mycobacterium avium and Mycobacterium intracellulare isolated in Puerto Rico from clinical samples by the use of a non-radioactive DNA probe.

    PubMed

    García, M T; Peña, I; Zlotnik, H

    1994-06-01

    The Mycobacterium avium complex (MAC), especially M. avium, is an important opportunistic pathogen of AIDS patients in the United States. In Puerto Rico, the incidence of infections caused by MAC has not been determined. This is due, in part, to the difficulties associated to the microbiological identification of the microorganisms. In this work, a commercially available kit (AccuProbe, Gen-Probe, Inc., San Diego, CA) utilizing a DNA probe complementary to rRNA of M. avium and M. intracellulare was used to identify seventeen MAC strains and one unknown atypical mycobacterium recovered in culture in Puerto Rico from clinical samples. The results obtained revealed that M. avium was the predominant species recovered (83% of isolates tested). Only two cultures were identified as M. intracellulare. The unknown culture, which did not react with either probe, turned out to be M. gordonae. The probe tests not only are simple to perform, but provide cultural identification results in as little as two hours. This study, the first one of its kind in Puerto Rico, demonstrates that the nucleic acid probes for the cultural identification of M. avium and M. intracellulare offer the potential of providing a prompt diagnosis and much needed data on the epidemiology of MAC infections in Puerto Rico.

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

    PubMed

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

    2016-04-05

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

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

    PubMed Central

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

    1993-01-01

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

  5. Probing disorder in isometric pyrochlore and related complex oxides

    DOE PAGES

    Shamblin, Jacob; Feygenson, Mikhail; Neuefeind, Joerg C; ...

    2016-02-29

    There has been an increased focus on understanding the energetics of structures that can accommodate unconventional ordering (e.g., disordered periodic arrays that appear at different length-scales). For example, the isometric pyrochlore structure, A2B2O7, forms a disordered, defect fluorite-structure, (A,B)4O7, or an aperiodic array, when exposed to extreme conditions]. The ability to accommodate disorder in its structure accounts for the tendency of some compositions to resist becoming aperiodic in high radiation fields or at high temperatures. Thus, these materials find application as host materials for immobilizing actinides, such as plutonium, fast ion conductors in solid oxide fuel cells, and thermal barriermore » coatings for gas turbine jet engines. Despite the importance of the disordering process, there has been only a limited understanding of the role of local ordering on the energetic landscape, mainly due to the use of techniques (i.e., X-ray/electron diffraction) that can only characterize the average structure over a large number unit cells and are insensitive to disorder on the oxygen sublattice. We have used neutron pair distribution functions (PDFs) to show that the disordered fluorite structure consists of a locally-ordered, orthorhombic structural unit that is repeated by a pseudo-translational symmetry, such that the ordered, orthorhombic and disordered, isometric arrays coexist at different length-scales. This disordering mechanism is a more general phenomenon in complex oxides, as PDF analysis has revealed that inversion in the isometric spinel structure, AB2O4 B(AB)O4, occurs by a similar process across the highly randomized B-site. This insight into order-disorder transformations induced intrinsically (chemical composition) or extrinsically (far from equilibrium conditions by high radiation fields) provides a new basis for understanding how modulated structures and correlated disorder at different length-scales affects the physical and

  6. Absorption spectroscopic probe to investigate the interaction between Nd(III) and calf-thymus DNA

    NASA Astrophysics Data System (ADS)

    Devi, Ch. Victory; Singh, N. Rajmuhon

    2011-03-01

    The interaction between Nd(III) and Calf Thymus DNA (CT-DNA) in physiological buffer (pH 7.4) has been studied using absorption spectroscopy involving 4f-4f transition spectra in different aquated organic solvents. Complexation with CT-DNA is indicated by the changes in absorption intensity following the subsequent changes in the oscillator strengths of different 4f-4f bands and Judd-Ofelt intensity ( Tλ) parameters. The other spectral parameters namely Slator-Condon ( Fk's), nephelauxetic effect ( β), bonding ( b1/2) and percent covalency ( δ) parameters are computed to correlate with the binding of Nd(III) with DNA. The absorption spectra of Nd(III) exhibited hyperchromism and red shift in the presence of DNA. The binding constant, Kb has been determined by absorption measurement. The relative viscosity of DNA decreased with the addition of Nd(III). Thermodynamic parameters have been calculated according to relevant absorption data and Van't Hoff equation. The characterisation of bonding mode has been studied in detail. The results suggested that the major interaction mode between Nd(III) and DNA was external electrostatic binding.

  7. Direct observation of DNA threading in flap endonuclease complexes

    PubMed Central

    AlMalki, Faizah A; Flemming, Claudia S; Zhang, Jing; Feng, Min; Sedelnikova, Svetlana E; Ceska, Tom; Rafferty, John B; Sayers, Jon R; Artymiuk, Peter J

    2016-01-01

    Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “fly-casting, thread, bend and barb” mechanism. PMID:27273516

  8. DNA Modified Fe3O4@Au Magnetic Nanoparticles as Selective Probes for Simultaneous Detection of Heavy Metal Ions.

    PubMed

    Miao, Peng; Tang, Yuguo; Wang, Lei

    2017-02-01

    Driven by the urgent need to detect trace heavy metal ions in various real water samples, this article demonstrates for the first time an electrochemical biosensor based on DNA modified Fe3O4@Au magnetic nanoparticles (NPs). Three DNA probes are designed to contain certain mismatched base pairs. One is thiolated and modified on the surface of Fe3O4@Au NPs (DNA 1). The other two probes (DNA 2 and 3) are labeled with two independent electrochemical species. Stable structures of cytosine-Ag(+)-cytosine and thymine-Hg(2+)-thymine formed in the presence of Ag(+) and Hg(2+) can assist the hybridization of DNA 1/DNA 2 and DNA 1/DNA 3, which locate corresponding electrochemical species onto the surface of the magnetic NPs. The achieved nanocomposites are then used as selective electrochemical probes for the detection of heavy metal ions by recording the square wave voltammetry signals. Simultaneous detection of Ag(+) and Hg(2+) is demonstrated without significant interference, and their individual high sensitivities are fundamentally preserved, which meet the requirements of U.S. Environmental Protection Agency (USEPA). Furthermore, the proposed method has been challenged by various real water samples. The results confirm the DNA modified magnetic NPs based sensing method may have potential applications for the monitoring of heavy metal ions in real sample analysis.

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

    SciTech Connect

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

    2014-09-03

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

  10. HUBBLE PROBES THE COMPLEX HISTORY OF A DYING STAR

    NASA Technical Reports Server (NTRS)

    2002-01-01

    This NASA Hubble Space Telescope image shows one of the most complex planetary nebulae ever seen, NGC 6543, nicknamed the 'Cat's Eye Nebula.' Hubble reveals surprisingly intricate structures including concentric gas shells, jets of high-speed gas and unusual shock-induced knots of gas. Estimated to be 1,000 years old, the nebula is a visual 'fossil record' of the dynamics and late evolution of a dying star. A preliminary interpretation suggests that the star might be a double-star system. The dynamical effects of two stars orbiting one another most easily explains the intricate structures, which are much more complicated than features seen in most planetary nebulae. (The two stars are too close together to be individually resolved by Hubble, and instead, appear as a single point of light at the center of the nebula.) According to this model, a fast 'stellar wind' of gas blown off the central star created the elongated shell of dense, glowing gas. This structure is embedded inside two larger lobes of gas blown off the star at an earlier phase. These lobes are 'pinched' by a ring of denser gas, presumably ejected along the orbital plane of the binary companion. The suspected companion star also might be responsible for a pair of high-speed jets of gas that lie at right angles to this equatorial ring. If the companion were pulling in material from a neighboring star, jets escaping along the companion's rotation axis could be produced. These jets would explain several puzzling features along the periphery of the gas lobes. Like a stream of water hitting a sand pile, the jets compress gas ahead of them, creating the 'curlicue' features and bright arcs near the outer edge of the lobes. The twin jets are now pointing in different directions than these features. This suggests the jets are wobbling, or precessing, and turning on and off episodically. The image was taken with the Wide Field Planetary Camera-2 on September 18, 1994. NGC 6543 is 3,000 light-years away in the

  11. Dissecting the effect of anions on Hg2+ detection using a FRET based DNA probe.

    PubMed

    Kiy, Mehmet Murat; Zaki, Ahmed; Menhaj, Arsalsan Beg; Samadi, Azadeh; Liu, Juewen

    2012-08-07

    Many biosensors have been developed to detect Hg(2+) using thymine-rich DNA. While sensor response to various cations is often studied to demonstrate selectivity, the effect of anions has been largely overlooked. Anions may compete with DNA for metal binding and thus produce a false negative result. Anions cannot be added alone; the cation part of a salt may cause DNA compaction and other effects, obscuring the role of anions. We find that the sensitivity of a FRET-based Hg(2+) probe is independent of Na(+) concentration. Therefore, by using various sodium salts, any change in sensitivity can be attributed solely to the effect of anions. Halide salts, sulfides, and amines are strong inhibitors; anions containing oxo or hydroxyl groups (e.g. nitrate, sulfate, phosphate, carbonate, acetate, and citrate) do not interfere with Hg(2+) detection even at 100 mM concentration. Mercury hydrolysis and its diffusion into polypropylene containers can also strongly affect the detection results. We conclude that thymine-rich DNA should be useful for Hg(2+) detection in many environmental water samples.

  12. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  13. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes

    NASA Astrophysics Data System (ADS)

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  14. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes.

    PubMed

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  15. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    PubMed

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  16. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

    PubMed

    Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

    2016-12-15

    The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

  17. Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

    SciTech Connect

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso

    2010-10-22

    Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

  18. A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

    PubMed Central

    Krasnoslobodtsev, Alexey V.; Zhang, Yuliang; Viazovkina, Ekaterina; Gall, Alexander; Bertagni, Chad; Lyubchenko, Yuri L.

    2015-01-01

    Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment. PMID:25954890

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

    PubMed

    Rikke, B A; Hardies, S C

    1991-12-01

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

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

    SciTech Connect

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

    1986-10-01

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

  1. Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection

    NASA Astrophysics Data System (ADS)

    Lee, Hyungbeen; Lee, Sang Won; Lee, Gyudo; Lee, Wonseok; Lee, Jeong Hoon; Hwang, Kyo Seon; Yang, Jaemoon; Lee, Sang Woo; Yoon, Dae Sung

    2016-07-01

    Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities.Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a

  2. Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level.

    PubMed

    Shin, Yong Kook; Jeong, Seung-U; Yoo, So Young; Kim, Mi-Kwang; Kim, Hwa-Sook; Kim, Byung-Ock; Kim, Do Kyung; Hwang, Ho-Keel; Kook, Joong-Ki

    2004-01-01

    Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.

  3. DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

    DOE PAGES

    Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

    2016-03-09

    The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

  4. DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

    SciTech Connect

    Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; Johnson, Reid C.; Leng, Fenfei

    2016-03-09

    The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

  5. Tuning the cellular uptake properties of luminescent heterobimetallic iridium(III)-ruthenium(II) DNA imaging probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; Turton, David; Adams, Harry; Roseveare, Thomas M; Smythe, Carl; Su, Xiaodi; Thomas, Jim A

    2014-10-20

    The synthesis of two new luminescent dinuclear Ir(III)-Ru(II) complexes containing tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir(III) moieties, these complexes display good water solubility, allowing the first cell-based study on Ir(III)-Ru(II) bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru(II) excited state and both complexes display significant in vitro DNA-binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA-imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2-(4-fluorophenyl)pyridine ligands around its Ir(III) centre is enhanced in comparison to the non-fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition-metal bioprobes.

  6. Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

    PubMed Central

    Lovell, C R; Hui, Y

    1991-01-01

    The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed. Images PMID:1768134

  7. Orientation of DNA Minicircles Balances Density and Topological Complexity in Kinetoplast DNA

    PubMed Central

    Diao, Yuanan; Rodriguez, Victor; Klingbeil, Michele; Arsuaga, Javier

    2015-01-01

    Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network. PMID:26110537

  8. DNA Hybridization Probe for Use in Determining Restricted Nodulation among Bradyrhizobium japonicum Serocluster 123 Field Isolates

    PubMed Central

    Sadowsky, Michael J.; Cregan, Perry B.; Keyser, Harold H.

    1990-01-01

    Several soybean plant introduction (PI) genotypes have recently been described which restrict nodulation of Bradyrhizobium japonicum serocluster 123 in an apparently serogroup-specific manner. While PI 371607 restricts nodulation of strains in serogroup 123 and some in serogroup 127, those in serogroup 129 are not restricted. When DNA regions within and around the B. japonicum I-110 common nodulation genes were used as probes to genomic DNA from the serogroup strains USDA 123, USDA 127, and USDA 129, several of the probes differentially hybridized to the nodulation-restricted and -unrestricted strains. One of the gene regions, cloned in plasmid pMJS12, was subsequently shown to hybridize to 4.6-kilobase EcoRI fragments from DNAs from nodulation-restricted strains and to larger fragments in nodulation-unrestricted strains. To determine if the different hybridization patterns could be used to predict nodulation restriction, we hybridized pMJS12 to EcoRI-digested genomic DNAs from uncharacterized serocluster 123 field isolates. Of the 36 strains examined, 15 were found to have single, major, 4.6-kilobase hybridizing EcoRI fragments. When tested for nodulation, 80% (12 of 15) of the strains were correctly predicted to be restricted for nodulation of the PI genotypes. In addition, hybridization patterns obtained with pMJS12 and nodulation phenotypes on PI 371607 indicated that there are at least three types of serogroup 127 strains. Our results suggest that the pMJS12 gene probe may be useful in selecting compatible host-strain combinations and in determining the suitability of field sites for the placement of soybean genotypes containing restrictive nodulation alleles. Images PMID:16348217

  9. A Simple and Fast Semiautomatic Procedure for the Atomistic Modeling of Complex DNA Polyhedra.

    PubMed

    Alves, Cassio; Iacovelli, Federico; Falconi, Mattia; Cardamone, Francesca; Morozzo Della Rocca, Blasco; de Oliveira, Cristiano L P; Desideri, Alessandro

    2016-05-23

    A semiautomatic procedure to build complex atomistic covalently linked DNA nanocages has been implemented in a user-friendly, free, and fast program. As a test set, seven different truncated DNA polyhedra, composed by B-DNA double helices connected through short single-stranded linkers, have been generated. The atomistic structures, including a tetrahedron, a cube, an octahedron, a dodecahedron, a triangular prism, a pentagonal prism, and a hexagonal prism, have been probed through classical molecular dynamics and analyzed to evaluate their structural and dynamical properties and to highlight possible building faults. The analysis of the simulated trajectories also allows us to investigate the role of the different geometries in defining nanocages stability and flexibility. The data indicate that the cages are stable and that their structural and dynamical parameters measured along the trajectories are slightly affected by the different geometries. These results demonstrate that the constraints imposed by the covalent links induce an almost identical conformational variability independently of the three-dimensional geometry and that the program presented here is a reliable and valid tool to engineer DNA nanostructures.

  10. Enantiopure copper(II) complex of natural product rosin derivative: DNA binding, DNA cleavage and cytotoxicity.

    PubMed

    Fei, Bao-Li; Yin, Bin; Li, Dong-Dong; Xu, Wu-Shuang; Lu, Yang

    2016-12-01

    To develop chiral anticancer drug candidates for molecular target DNA, the synthesis and characterization of a novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1) was carried out. The coordination geometry of the copper center is a distorted square-planar arrangement. The interactions of 1 and 2 with salmon sperm DNA were investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. All the results reveal that 1 and 2 interacted with DNA through intercalation and 2 exhibited a higher DNA binding ability. Further, 1 and 2 could cleave supercoiled pBR322 DNA by single strand and 2 displayed stronger cleavage ability in the presence of ascorbic acid. In vitro cytotoxicity of 1 and 2 against HeLa, SiHa, HepG-2 and A431 cancer cell lines was studied using CCK-8 assay. The results indicate that 2 had a superior cytotoxicity than 1 and the widely used drug cisplatin under identical conditions. Flow cytometry analysis demonstrates 2 produced death of HeLa cancer cells through an apoptotic pathway. Cell cycle analysis shows that 2 mainly arrested HeLa cells at the S phase. A novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1), based on natural product rosin has been synthesized. 2 has the potential to act as effective anticancer drug.

  11. Near IR-emitting DNA-probes exploiting stepwise energy transfer processes.

    PubMed

    Bodi, Andras; Borbas, K Eszter; Bruce, James I

    2007-10-10

    The synthesis and characterisation of two new cyclen-based near IR-emitting lanthanide complexes is reported; the lanthanides are sensitised by rhodamine, which in turn is excited by energy transfer from a coumarin 2 moiety. The three lumophores function as an energy transfer cascade spanning the UV-visible-near IR region of the spectrum, resulting in large Stokes shifts. Double stranded DNA selectively switches one of the two energy transfer processes off, enabling luminescent DNA-sensing in the near IR region. The regioselective di-alkylation of the cyclen scaffold is explained with the help of DFT calculations.

  12. The FACT complex promotes avian leukosis virus DNA integration.

    PubMed

    Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

    2017-01-25

    All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. Here, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-LTR circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

  13. Facile construction of a highly sensitive DNA biosensor by in-situ assembly of electro-active tags on hairpin-structured probe fragment

    PubMed Central

    Wang, Qingxiang; Gao, Feng; Ni, Jiancong; Liao, Xiaolei; Zhang, Xuan; Lin, Zhenyu

    2016-01-01

    An ultrasensitive DNA biosensor has been developed through in-situ labeling of electroactive melamine-Cu2+ complex (Mel-Cu2+) on the end of hairpin-like probe using gold nanoparticles (AuNPs) as the signal amplification platform. The 3′-thiolated hairpin-like probe was first immobilized to the gold electrode surface by the Au-S bond. The AuNPs were then tethered on the free 5′-end of the immobilized probe via the special affinity between Au and the modified -NH2. Followed by, the Mel and Cu2+ were assembled on the AuNPs surface through Au-N bond and Cu2+-N bond, respectively. Due to the surface area and electrocatalytic effects of the AuNPs, the loading amount and electron transfer kinetic of the Mel-Cu2+ were enhanced greatly, resulting in significantly enhanced electrochemical response of the developed biosensor. Compared with the synthesis process of conventional electroactive probe DNA accomplished by homogeneous method, the method presented in this work is more reagent- and time-saving. The proposed biosensor showed high selectivity, wide linear range and low detection limit. This novel strategy could also be extended to the other bioanalysis platforms such as immunosensors and aptasensors. PMID:26931160

  14. Measuring complexity, nonextensivity and chaos in the DNA sequence of the Major Histocompatibility Complex

    NASA Astrophysics Data System (ADS)

    Pavlos, G. P.; Karakatsanis, L. P.; Iliopoulos, A. C.; Pavlos, E. G.; Xenakis, M. N.; Clark, Peter; Duke, Jamie; Monos, D. S.

    2015-11-01

    We analyze 4 Mb sequences of the Major Histocompatibility Complex (MHC), which is a DNA segment on chromosome 6 with high gene density, controlling many immunological functions and associated with many diseases. The analysis is based on modern theoretical and mathematical tools of complexity theory, such as nonlinear time series analysis and Tsallis non-extensive statistics. The results revealed that the DNA complexity and self-organization can be related to fractional dynamical nonlinear processes with low-dimensional deterministic chaotic and non-extensive statistical character, which generate the DNA sequences under the extremization of Tsallis q-entropy principle. While it still remains an open question as to whether the DNA walk is a fractional Brownian motion (FBM), a static anomalous diffusion process or a non-Gaussian dynamical fractional anomalous diffusion process, the results of this study testify for the latter, providing also a possible explanation for the previously observed long-range power law correlations of nucleotides, as well as the long-range correlation properties of coding and non-coding sequences present in DNA sequences.

  15. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    PubMed

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.

  16. Single-probe multistate detection of DNA via aggregation-induced emission on a graphene oxide platform.

    PubMed

    Tyagi, Abhishek; Chu, Kin Leung; Abidi, Irfan Haider; Cagang, Aldrine Abenoja; Zhang, Qicheng; Leung, Nelson L C; Zhao, Engui; Tang, Ben Zhong; Luo, Zhengtang

    2017-03-01

    Graphene and graphene oxides (GO), or their reduced forms, have been introduced in a variety of biosensing platforms and have exhibited enhanced performance levels in these forms. We herein report a DNA sensing platform consisting of aggregation-induced emission (AIE) molecules and complementary DNA (comDNA) adsorbed on GO. We experimentally turned the AIE molecule on and off by adjusting its distance, which correlates with DNA structures as shown in our computational results, from the GO sheet, which quenches depending on its distance from the graphene plane. The changes in florescence are reproducible, which demonstrates the probe's ability to identify the binding state of the DNA. Our molecular dynamics simulation results reveal strong π-π interactions between single-strand DNA (ssDNA) and GO, which enable the ssDNA molecule to move closer to the graphene oxide. This reduces the center of mass and binding free energies in the simulation. When hybridized with comDNA, the increased distance, evidenced by the reduced interaction, eliminates the quenching effect and turns on the AIE molecule. Our protocol use of the AIE molecule as a probe thus avoids the complicated steps involved in covalent functionalization and allows the rapid and label-free detection of DNA molecules.

  17. DNA-METAFECTENE PRO complexation: a physical chemistry study.

    PubMed

    Alatorre-Meda, Manuel; González-Pérez, Alfredo; Rodríguez, Julio R

    2010-07-21

    Complexes formed between cationic liposomes and DNA (also known as lipoplexes or genosomes) have proven, for years now, to be a suitable option for gene delivery to cells, transfection, however, some aspects regarding the liposome-DNA interaction mechanism and complex stability remain still unclear. This work aims to improve the understanding of the poorly defined mechanisms and structural conformation associated with the interaction of METAFECTENE PRO (MEP), a commercial liposomal transfection reagent, with poly-anion DNA at mass ratios around the mass ratio recommended for transfection (L/D congruent with 700). A physical chemistry characterization was conducted at a pH of 6.5 and at a temperature of 25 degrees C by means of dynamic light scattering (DLS), electrophoretic mobility (zeta-potential), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Five parameters important for transfection were determined for the lipoplexes: (i) the hydrodynamic radius, R(H), (ii) the stability with time, (iii) the mass ratio of at which both moieties start to interact, (L/D)(i), (iv) the overall charge, and (v) the morphology. Results in ensemble point to a "beads on a string" conformation, with the lipoplex formation occurring well below isoneutrality from (L/D)(i) congruent with 600. The lipoplexes were found to be stable within at least seven days presenting an average R(H) of 135 nm.

  18. Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

    PubMed

    Zheng, Wenjun

    2017-02-01

    In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals

  19. Quantum-dot-labeled DNA probes for fluorescence in situ hybridization (FISH) in the microorganism Escherichia coli.

    PubMed

    Wu, Sheng-Mei; Zhao, Xiang; Zhang, Zhi-Ling; Xie, Hai-Yan; Tian, Zhi-Quan; Peng, Jun; Lu, Zhe-Xue; Pang, Dai-Wen; Xie, Zhi-Xiong

    2006-05-12

    Semiconductor quantum dots (QDs) as a kind of nonisotopic biological labeling material have many unique fluorescent properties relative to conventional organic dyes and fluorescent proteins, such as composition- and size-dependent absorption and emission, a broad absorption spectrum, photostability, and single-dot sensitivity. These properties make them a promising stable and sensitive label, which can be used for long-term fluorescent tracking and subcellular location of genes and proteins. Here, a simple approach for the construction of QD-labeled DNA probes was developed by attaching thiol-ssDNA to QDs via a metal-thiol bond. The as-prepared QD-labeled DNA probes had high dispersivity, bioactivity, and specificity for hybridization. Based on such a kind of probe with a sequence complementary to multiple clone sites in plasmid pUC18, fluorescence in situ hybridization of the tiny bacterium Escherichia coli has been realized for the first time.

  20. Padlock probe-mediated qRT-PCR for DNA computing answer determination

    PubMed Central

    Xiong, Fusheng; Frasch, Wayne D.

    2011-01-01

    Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology. PMID:21691417

  1. Probing the active site tightness of DNA polymerase in subangstrom increments.

    PubMed

    Kim, Tae Woo; Delaney, James C; Essigmann, John M; Kool, Eric T

    2005-11-01

    We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.

  2. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing.

    PubMed

    Jiang, Longfei; Song, Mengke; Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.

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

    PubMed

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

    2001-11-01

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

  4. Identification of the autotrophic denitrifying community in nitrate removal reactors by DNA-stable isotope probing.

    PubMed

    Xing, Wei; Li, Jinlong; Cong, Yuan; Gao, Wei; Jia, Zhongjun; Li, Desheng

    2017-04-01

    Autotrophic denitrification has attracted increasing attention for wastewater with insufficient organic carbon sources. Nevertheless, in situ identification of autotrophic denitrifying communities in reactors remains challenging. Here, a process combining micro-electrolysis and autotrophic denitrification with high nitrate removal efficiency was presented. Two batch reactors were fed organic-free nitrate influent, with H(13)CO3(-) and H(12)CO3(-) as inorganic carbon sources. DNA-based stable-isotope probing (DNA-SIP) was used to obtain molecular evidence for autotrophic denitrifying communities. The results showed that the nirS gene was strongly labeled by H(13)CO3(-), demonstrating that the inorganic carbon source was assimilated by autotrophic denitrifiers. High-throughput sequencing and clone library analysis identified Thiobacillus-like bacteria as the most dominant autotrophic denitrifiers. However, 88% of nirS genes cloned from the (13)C-labeled "heavy" DNA fraction showed low similarity with all culturable denitrifiers. These findings provided functional and taxonomical identification of autotrophic denitrifying communities, facilitating application of autotrophic denitrification process for wastewater treatment.

  5. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing

    PubMed Central

    Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ. PMID:26098417

  6. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between L-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH = 4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that L-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of L-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  7. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe.

    PubMed

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between l-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH=4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that l-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of l-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  8. Surface plasmon resonance technique for directly probing the interaction of DNA and graphene oxide and ultra-sensitive biosensing.

    PubMed

    Xue, Tianyu; Cui, Xiaoqiang; Guan, Weiming; Wang, Qiyu; Liu, Chang; Wang, Haitao; Qi, Kun; Singh, D J; Zheng, Weitao

    2014-08-15

    The binding of DNA with graphene oxide (GO) is important for applications in disease diagnosis, genetic screening, and drug discovery. The standard assay methods are mainly limited to indirect observation via fluorescence labeling. Here we report the use of surface plasmon resonance for direct sensing of DNA/GO binding. We show that this can be used for ultra-sensitive detection of single-stranded DNA (ssDNA). Furthermore, the results provide a more direct probe of DNA/GO binding abilities and confirm that hydrogen bonding plays a key role in the interaction between GO and ssDNA. This enables to a novel biosensor for highly sensitive and selective detection of ssDNA based on indirect competitive inhibition assay (ICIA). We report development of such a sensor with a linear dynamic range of 10(-14)-10(-6)M, a detection limit of 10fM and a high level of stability during repeated regeneration.

  9. Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.

    PubMed

    Blosser, Timothy R; Loeff, Luuk; Westra, Edze R; Vlot, Marnix; Künne, Tim; Sobota, Małgorzata; Dekker, Cees; Brouns, Stan J J; Joo, Chirlmin

    2015-04-02

    Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.

  10. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    SciTech Connect

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. )

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

  11. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

    SciTech Connect

    Changela, A.; Digate, R.J.; Mondragon, A.

    2010-03-05

    A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

  12. Design and characterization of a novel lipid-DNA complex that resists serum-induced destabilization.

    PubMed

    Lian, Tianshun; Ho, Rodney J Y

    2003-12-01

    Ineffectiveness of cationic lipids to enhance DNA transfection has been attributed to serum-mediated dissociation and perhaps complement activation of lipid-DNA complexes. To overcome these problems, we have developed a novel lipid-DNA complex that greatly reduces serum-mediated dissociation. The complexes were prepared by mixing cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphatidyl-ethanolamine and DNA in ethanolic (20% v/v ethanol) solution containing 5% sucrose followed by dehydration via rotating evaporation. Upon hydration in H(2)O, the lipid-DNA complexes [ethanol-dried lipid-DNA (EDL) complexes] were formed. The complexes exhibit a low positive zeta potential and enhanced transfection efficiency in contrast to the suppressed efficiency detected with admixed lipid-DNA complexes in the presence of serum across several cell lines. This result may be attributed to the inability of serum to dissociate DNA from lipids in EDL complexes. Using displacement of ethidium bromide intercalation analysis, we found that in serum, only 50% of DNA was exposed in the EDL complexes, compared with 100% in the admixed lipid-DNA complexes. The EDL complexes also showed increased resistance to DNase digestion in the presence of negatively charged lipid, while reducing complement activation in serum. The EDL complexes may improve the transfection activity of lipid-DNA complexes in serum and, perhaps, in vivo.

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

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

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

  14. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    PubMed

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

  15. Accuracy enhancement of wideband complex permittivity measured by an open-ended coaxial probe

    NASA Astrophysics Data System (ADS)

    Jung, Ji-Hyun; Cho, Jae-Hyoung; Kim, Se-Yun

    2016-01-01

    When the wideband complex permittivity of a liquid solution was measured by an open-ended coaxial probe, its accuracy was inherently degraded due to an inexact conversion model compared with the uncertainty of the associated vector network analyzer. In this paper, the accuracy of the converted wideband complex permittivity is evaluated indirectly and then enhanced significantly. Firstly, the measured wideband complex permittivity is fitted by a dispersive permittivity profile in the Debye formula. Secondly, the new reflection coefficients are calculated numerically by applying the dispersive permittivity profile to a 2D finite-difference time-domain model of our probe. Thirdly, the inaccuracy of the fitted complex permittivity profile is quantified indirectly by the root mean square (RMS) error of the numerically calculated reflection coefficients in comparison with the originally measured reflection coefficients. Finally, four parameters of the Debye formula involved in the initially converted complex permittivity profile were calibrated iteratively until the RMS error of the repeatedly calculated reflection coefficients could be minimized. The validity of the above calibration procedure is assured numerically for a given wideband complex permittivity profile. In the case of our actual measurement of the fabricated cancer-equivalent solution, an unknown complex permittivity profile, the RMS error of the numerically calculated reflection coefficients could be reduced from 0.0416 to 0.0093. This calibration results in both relative dielectric constant and conductivity profiles being gradually enhanced as the frequency increases up to 5000 MHz.

  16. Variation of entropic elasticity of DNA-Psoralen complex under UV light

    NASA Astrophysics Data System (ADS)

    Rocha, M. S.; Mesquita, O. N.

    2005-08-01

    We measure the entropic elasticity of a single λ-DNA molecule and of a single DNA-Psoralen complex by doing stretching experiments with an optical tweezers. Psoralen is a photosensitive drug used in the treatment of many skin diseases, by impeding DNA replication. Psolaren intercalates the DNA and can form crosslinks with pyrimidine basis in opposite strands of DNA, when illuminated with UVA light. As crosslinks form the persistence length of the complex increases, indicating an increase in rigidity of the complex. We study the kinetics of DNA-Psoralen crosslink formation via changes in entropic elasticity of the complex.

  17. Endovascular Repair of Complex Aortic Aneurysms: Intravascular Ultrasound Guidance with an Intracardiac Probe

    SciTech Connect

    Zanchetta, Mario Rigatelli, Gianluca; Pedon, Luigi; Zennaro, Marco; Ronsivalle, Salvatore; Maiolino, Pietro

    2003-09-15

    To assess the accuracy and efficacy of intravascular ultrasound guidance obtained by an intracardiac ultrasound probe during complex aortic endografting. Between November 1999 and July 2002, 19 patients (5 female, 14 male; mean age 73.5 {+-} 2.1 years) underwent endovascular repair of thoracic (n = 10), complex abdominal (n = 6) and concomitant thoraco-abdominal (n = 3) aortic aneurysm. The most suitable size and configuration of the stent-graft were chosen on the basis of preoperative computed tomographic angiography (CTA) or magnetic resonance angiography (MRA). Intraoperative intravascular ultrasound imaging was obtained using a 9 Fr, 9 MHz intracardiac echocardiography (ICE) probe, 110 cm in length, inserted through a 10 Fr precurved long sheath. The endografts were deployed as planned by CTA or MRA. Before stent-graft deployment, the ICE probe allowed us to view the posterior aortic arch and descending thoraco-abdominal aorta without position-related artifacts, and to identify both sites of stent-graft positioning. After stent-graft deployment, the ICE probe allowed us to detect the need for additional modular components to internally reline the aorta in 11 patients, and to discover 2 incomplete graft expansions subsequently treated with adjunctive balloon angioplasty. In 1 patient, the ICE probe supported the decision that the patient was ineligible for the endovascular exclusion procedure. The ICE probe provides accurate information on the anatomy of the posterior aortic arch and thoracic and abdominal aortic aneurysms and a rapid identification of attachment sites and stent-graft pathology, allowing refinement and improvement of the endovascular strategy.

  18. Mercury-199 NMR of the Metal Receptor Site in MerR and Its Protein-DNA Complex

    NASA Astrophysics Data System (ADS)

    Utschig, Lisa M.; Bryson, James W.; O'Halloran, Thomas V.

    1995-04-01

    Structural insights have been provided by mercury-199 nuclear magnetic resonance (NMR) into the metal receptor site of the MerR metalloregulatory protein alone and in a complex with the regulatory target, DNA. The one- and two-dimensional NMR data are consistent with a trigonal planar Hg-thiolate coordination environment consisting only of Cys side chains and resolve structural aspects of both metal ion recognition and the allosteric mechanism. These studies establish 199Hg NMR techniques as useful probes of the metal coordination environment of regulatory proteins, copper enzymes, and zinc transcription factor complexes as large as 50 kilodaltons.

  19. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex

    PubMed Central

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  20. Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

    PubMed

    Paradh, A D; Hill, A E; Mitchell, W J

    2014-01-01

    DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

  1. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    PubMed

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  2. Potential use of buccal smears for rapid diagnosis of autosomal trisomy or chromosomal sex in newborn infants using DNA probes

    SciTech Connect

    Harris, C.; Clark, K.; Lazarski, K.; Wilkerson, C.; Meisner, L. |

    1994-12-01

    Buccal smears from 3 women and 1 man were probed with alpha satellite DNA probes for chromosomes 8, 18, X, and Y. Buccal smears were also collected from an adolescent phenotypic female with uterine agenesis, as well as from newborn infants with suspected trisomy 18 and trisomy 21. The clinical cases were confirmed with conventional cytogenetic studies of peripheral lymphocytes. Overall probe efficiency at detecting expected chromosome number in interphase cells was found to be 71% {+-} 6.8%. Higher than expected n-1 signal numbers may be due to karyopyknotic intermediate epithelial cells present in all collected samples. Overall probe efficiency was found to be consistent using alpha satellite and cosmid probes, both of which accurately reflected the modal copy number of the target chromosomes. False trisomy was less than 1%. This study suggests DNA probes can be used in buccal smears for rapid diagnosis of trisomies and chromosomal sex in newborns, but because of high rates of false hydropoploid signals, probed buccal smear specimens may not be accurate at diagnosing mosaicism. 9 refs., 2 figs., 1 tab.

  3. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    PubMed Central

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  4. Studying complex fluids and crystal formation using Dynamic Light Scattering and Optical Probe Diffusion

    NASA Astrophysics Data System (ADS)

    Streletzky, Kiril A.

    2004-10-01

    Dynamic light scattering (DLS) spectroscopy provides non-destructive analytic technique for probing structure and dynamics of polymer solutions, colloids, micellar systems, liquid crystals, and, in some cases, observation of nucleation and crystal growth. The method of optical probe diffusion uses DLS to measure transport of dilute mesoscopic probes in a complex fluid solution. If tracer particles are the dominant scatterers in solution, their diffusion provides important information for inferring physical properties of the complex fluid. In situ nature of DLS allows non-invasive studies of different chemical and physical processes such as polymer and colloidal diffusion, gelation, micellar aggregation and dynamics. DLS is also useful for studying critical behavior of liquid crystals and for observing crystallization from clear solutions. The highlights of our results obtained using DLS and optical probe diffusion are presented. These examples include: a) research on polymer dynamics in dilute and concentrated solutions of a neutral HPC polymer; b) project on aggregation and transport of spherical Tx-100 micelles; c) study of crystallization of molecular sieve zeolite NaA.

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

    PubMed

    Do, Youngjin; Kim, Jongsung

    2016-03-01

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

  6. A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

    PubMed

    Yan, Zhijiang; Delannoy, Mathieu; Ling, Chen; Daee, Danielle; Osman, Fekret; Muniandy, Parameswary A; Shen, Xi; Oostra, Anneke B; Du, Hansen; Steltenpool, Jurgen; Lin, Ti; Schuster, Beatrice; Décaillet, Chantal; Stasiak, Andrzej; Stasiak, Alicja Z; Stone, Stacie; Hoatlin, Maureen E; Schindler, Detlev; Woodcock, Christopher L; Joenje, Hans; Sen, Ranjan; de Winter, Johan P; Li, Lei; Seidman, Michael M; Whitby, Matthew C; Myung, Kyungjae; Constantinou, Angelos; Wang, Weidong

    2010-03-26

    FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

  7. Chemical Methylation of RNA and DNA Viral Genomes as a Probe of In Situ Structure

    PubMed Central

    Yamakawa, Minoru; Shatkin, Aaron J.; Furuichi, Yasuhiro

    1981-01-01

    We used [methyl-3H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for φX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [methyl-3H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, φX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation. PMID:6172596

  8. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    PubMed Central

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R.G.; Stach, E.A.; Frenkel, A.I.

    2015-01-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes. PMID:26119246

  9. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    SciTech Connect

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-29

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. Lastly, this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  10. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    DOE PAGES

    Li, Y.; Zakharov, D.; Zhao, S.; ...

    2015-06-29

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. Lastly,more » this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.« less

  11. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    NASA Astrophysics Data System (ADS)

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction--ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  12. A DNA topoisomerase VI-like complex initiates meiotic recombination.

    PubMed

    Vrielynck, Nathalie; Chambon, Aurélie; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; De Muyt, Arnaud; Mézard, Christine; Mayer, Claudine; Grelon, Mathilde

    2016-02-26

    The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure.

  13. Ultra-sensitive complex as phosphorescence probe for the determination of trace protein.

    PubMed

    Liu, Jia-Ming; Jiang, Shu-Lian; Cui, Ma-Lin; Jiao, Li; Zhang, Li-Hong; Zheng, Zhi-Yong

    2013-02-01

    β-CD-HMTA-L-Tyr complex, formed in the host guest inclusion reaction carried out between host molecule β-cyclodextrin (β-CD) in β-CD-HMTA (HMTA is methenamine) and guest molecule L-tryptophan (L-Tyr), possessing the characteristic of room temperature phosphorescence (RTP). Bovine serum albumin (BSA) reacted with L-Tyr to form a complex of cage structure bringing in the sharply RTP signal quenching of L-Tyr. Based on the above facts, a new ultra-sensitive solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace protein has been established using β-CD-HMTA-L-Tyr complex as a phosphorescence probe. Under the optimum conditions, the linear range of this method was 0.0040-0.56 agspot(-1) with a detection limit (D.L.) as 0.92 zgspot(-1), and the regression equations of working curve was ΔI(p)=0.8239+162.5 m(BSA) (agspot(-1), n=8) with the correlation coefficient (r) of 0.9994. The relatively standard deviation (RSD) and the recovery of SSRTP were 4.8-3.3% and 96.7-102%, respectively, indicating that this method had good repeatability. The proposed phosphorescence probe has been applied in the detection of protein in real samples and the results agreed well with those obtained with SSRTP using methylene blue-sodium tetraphenylborate as phosphorescence probe. Meanwhile, the reaction mechanism for the determination of trace protein with β-CD-HMTA-L-Tyr complex as phosphorescence probe has been discussed.

  14. Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. )

    1989-03-07

    Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

  15. Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O⁶-methyl-G in DNA duplexes.

    PubMed

    Angelov, Todor; Dahlmann, Heidi A; Sturla, Shana J

    2013-10-15

    Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O(6)-G alkylation adducts. To optimize O(6)-Me-G-targeting probes, an understanding of how base pairs with O(6)-Me-G are stabilized is needed. In this study, we compared the ability of O(6)-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O(6)-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O(6)-G alkylation adducts in DNA.

  16. Surface-enhanced Raman scattering spectroscopy of topotecan-DNA complexes: Binding to DNA induces topotecan dimerization

    NASA Astrophysics Data System (ADS)

    Mochalov, K. E.; Strel'Tsov, S. A.; Ermishov, M. A.; Grokhovskii, S. L.; Zhuze, A. L.; Ustinova, O. A.; Sukhanova, A. V.; Nabiev, I. R.; Oleinikov, V. A.

    2002-09-01

    The interaction of topotecan (TPT), antitumor inhibitor of human DNA topoisomerase I, with calf thymus DNA was studied by surface-enhanced Raman scattering (SERS) spectroscopy. The SERS spectra of TPT are found to depend on its concentration in solution, which is associated with the dimerization of TPT. The spectral signatures of dimerization are identified. It is shown that binding to DNA induces the formation of TPT dimers. The formation of DNA-TPT-TPT-DNA complexes is considered as one of the possible mechanisms of human DNA topoisomerase I inhibition.

  17. Determining electron temperature for small spherical probes from network analyzer measurements of complex impedance

    NASA Astrophysics Data System (ADS)

    Walker, D. N.; Fernsler, R. F.; Blackwell, D. D.; Amatucci, W. E.

    2008-12-01

    In earlier work, using a network analyzer, it was shown that collisionless resistance (CR) exists in the sheath of a spherical probe when driven by a small rf signal. The CR is inversely proportional to the plasma density gradient at the location where the applied angular frequency equals the plasma frequency ωpe. Recently, efforts have concentrated on a study of the low-to-intermediate frequency response of the probe to the rf signal. At sufficiently low frequencies, the CR is beyond cutoff, i.e., below the plasma frequency at the surface of the probe. Since the electron density at the probe surface decreases as a function of applied (negative) bias, the CR will extend to lower frequencies as the magnitude of negative bias increases. Therefore to eliminate both CR and ion current contributions, the frequencies presently being considered are much greater than the ion plasma frequency, ωpi, but less than the plasma frequency, ωpe(r0), where r0 is the probe radius. It is shown that, in this frequency regime, the complex impedance measurements made with a network analyzer can be used to determine electron temperature. An overview of the theory is presented along with comparisons to data sets made using three stainless steel spherical probes of different sizes in different experimental environments and different plasma parameter regimes. The temperature measurements made by this method are compared to those made by conventional Langmuir probe sweeps; the method shown here requires no curve fitting as is the usual procedure with Langmuir probes when a Maxwell-Boltzmann electron distribution is assumed. The new method requires, however, a solution of the Poisson equation to determine the approximate sheath dimensions and integrals to determine approximate plasma and sheath inductances. The solution relies on the calculation of impedance for a spherical probe immersed in a collisionless plasma and is based on a simple circuit analogy for the plasma. Finally, the

  18. Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments

    PubMed Central

    Pan, Kailing; Li, Hongyu; Fan, Xiaoyan; Sun, Lixin; Zhang, Shujun; Gao, Yongqing

    2017-01-01

    In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays. PMID:28056074

  19. Comparative Analysis of Satellite DNA in the Drosophila melanogaster Species Complex

    PubMed Central

    Jagannathan, Madhav; Warsinger-Pepe, Natalie; Watase, George J.; Yamashita, Yukiko M.

    2016-01-01

    Satellite DNAs are highly repetitive sequences that account for the majority of constitutive heterochromatin in many eukaryotic genomes. It is widely recognized that sequences and locations of satellite DNAs are highly divergent even in closely related species, contributing to the hypothesis that satellite DNA differences may underlie speciation. However, due to its repetitive nature, the mapping of satellite DNAs has been mostly left out of recent genomics analyses, hampering the use of molecular genetics techniques to better understand their role in speciation and evolution. Satellite DNAs are most extensively and comprehensively mapped in Drosophila melanogaster, a species that is also an excellent model system with which to study speciation. Yet the lack of comprehensive knowledge regarding satellite DNA identity and location in its sibling species (D. simulans, D. mauritiana, and D. sechellia) has prevented the full utilization of D. melanogaster in studying speciation. To overcome this problem, we initiated the mapping of satellite DNAs on the genomes of the D. melanogaster species complex (D. melanogaster, D. simulans, D. mauritiana, and D. sechellia) using multi-color fluorescent in situ hybridization (FISH) probes. Our study confirms a striking divergence of satellite DNAs in the D. melanogaster species complex, even among the closely related species of the D. simulans clade (D. simulans, D. mauritiana, and D. sechellia), and suggests the presence of unidentified satellite sequences in these species. PMID:28007840

  20. Comparative Analysis of Satellite DNA in the Drosophila melanogaster Species Complex.

    PubMed

    Jagannathan, Madhav; Warsinger-Pepe, Natalie; Watase, George J; Yamashita, Yukiko M

    2017-02-09

    Satellite DNAs are highly repetitive sequences that account for the majority of constitutive heterochromatin in many eukaryotic genomes. It is widely recognized that sequences and locations of satellite DNAs are highly divergent even in closely related species, contributing to the hypothesis that satellite DNA differences may underlie speciation. However, due to its repetitive nature, the mapping of satellite DNAs has been mostly left out of recent genomics analyses, hampering the use of molecular genetics techniques to better understand their role in speciation and evolution. Satellite DNAs are most extensively and comprehensively mapped in Drosophila melanogaster, a species that is also an excellent model system with which to study speciation. Yet the lack of comprehensive knowledge regarding satellite DNA identity and location in its sibling species (D. simulans, D. mauritiana, and D. sechellia) has prevented the full utilization of D. melanogaster in studying speciation. To overcome this problem, we initiated the mapping of satellite DNAs on the genomes of the D. melanogaster species complex (D. melanogaster, D. simulans, D. mauritiana, and D. sechellia) using multi-color fluorescent in situ hybridization (FISH) probes. Our study confirms a striking divergence of satellite DNAs in the D. melanogaster species complex, even among the closely related species of the D. simulans clade (D. simulans, D. mauritiana, and D. sechellia), and suggests the presence of unidentified satellite sequences in these species.

  1. An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes

    PubMed Central

    Fagan, Rebecca L.; Wu, Meng; Chédin, Frédéric; Brenner, Charles

    2013-01-01

    DNA methyltransferase 1 (DNMT1) is the enzyme most responsible for epigenetic modification of human DNA and the intended target of approved cancer drugs such as 5-aza-cytidine and 5-aza-2′-deoxycytidine. 5-aza nucleosides have complex mechanisms of action that require incorporation into DNA, and covalent trapping and proteolysis of DNMT isozymes. Direct DNMT inhibitors are needed to refine understanding of the role of specific DNMT isozymes in cancer etiology and, potentially, to improve cancer prevention and treatment. Here, we developed a high throughput pipeline for identification of direct DNMT1 inhibitors. The components of this screen include an activated form of DNMT1, a restriction enzyme-coupled fluorigenic assay performed in 384 well plates with a z-factor of 0.66, a counter screen against the restriction enzyme, a screen to eliminate DNA intercalators, and a differential scanning fluorimetry assay to validate direct binders. Using the Microsource Spectrum collection of 2320 compounds, this screen identified nine compounds with dose responses ranging from 300 nM to 11 µM, representing at least two different pharmacophores with DNMT1 inhibitory activity. Seven of nine inhibitors identified exhibited two to four-fold selectivity for DNMT1 versus DNMT3A. PMID:24236046

  2. GENETIC DIVERSITY OF TYPHA LATIFOLIA (TYPHACEAE) AND THE IMPACT OF POLLUTANTS EXAMINED WITH TANDEM-REPETITIVE DNA PROBES

    EPA Science Inventory

    Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation w...

  3. Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

    PubMed

    Barcellona, Maria Luisa; Gammon, Seth; Hazlett, Theodore; Digman, Michelle A; Gratton, Enrico

    2004-11-01

    We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

  4. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes.

    PubMed

    Mecca, Carolina Z P; Fonseca, Fernando L A; Bagatin, Izilda A

    2016-11-05

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al(3+), Mg(2+), Zn(2+), and Cd(2+) salts (1-8); and characterized. The (1)H NMR spectra of complexes 1 and 5, containing Al(3+), were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

  5. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes

    NASA Astrophysics Data System (ADS)

    Mecca, Carolina Z. P.; Fonseca, Fernando L. A.; Bagatin, Izilda A.

    2016-11-01

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al3+, Mg2+, Zn2+, and Cd2+ salts (1-8); and characterized. The 1H NMR spectra of complexes 1 and 5, containing Al3+, were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

  6. Multicolor Gold-Silver Nano-Mushrooms as Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/miRNA Detection.

    PubMed

    Su, Jing; Wang, Dongfang; Nörbel, Lena; Shen, Jianlei; Zhao, Zhihan; Dou, Yanzhi; Peng, Tianhuan; Shi, Jiye; Mathur, Sanjay; Fan, Chunhai; Song, Shiping

    2017-02-21

    Uniform silver-containing metal nanostructures with strong and stable surface-enhanced Raman scattering (SERS) signals hold great promise for developing ultrasensitive probes for biodetection. Nevertheless, the direct synthesis of such ready-to-use nanoprobes remains extremely challenging. Herein we report a DNA-mediated gold-silver nanomushroom with interior nanogaps directly synthesized and used for multiplex and simultaneous SERS detection of various DNA and RNA targets. The DNA involved in the nanostructures can act as not only gap DNA (mediated DNA) but also probe DNA (hybridized DNA), and DNA's involvement enables the nanostructures to have the inherent ability to recognize DNA and RNA targets. Importantly, we were the first to establish a new method for the generation of multicolor SERS probes using two different strategies. First Raman-labeled alkanethiol probe DNA was assembled on gold nanoparticles, and second, thiol-containing Raman reporters were coassembled with the probe DNA. The ready-to-use probes also give great potential to develop ultrasensitive detection methods for various biological molecules.

  7. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, A.N.; Benson, S.C.

    1995-03-28

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figures.

  8. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, Alexander M.; Benson, Scott C.

    1999-01-01

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

  9. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, A.N.; Benson, S.C.

    1997-07-08

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figs.

  10. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, A.M.; Benson, S.C.

    1998-06-16

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figs.

  11. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, Alexander N.; Benson, Scott C.

    1995-01-01

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

  12. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, Alexander M.; Benson, Scott C.

    1998-01-01

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

  13. DNA complexes with dyes designed for energy transfer as fluorescent markers

    DOEpatents

    Glazer, Alexander N.; Benson, Scott C.

    1997-01-01

    Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

  14. Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

    PubMed Central

    Barcellona, M L; Gratton, E

    1996-01-01

    Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides. Images FIGURE 3 PMID:9172758

  15. FISH analysis of the arrangement of chromosomes in interphase nuclei using telomeric, centromeric, and DNA painting probes

    NASA Astrophysics Data System (ADS)

    Monajembashi, Shamci; Schmitt, Eberhard; Dittmar, Heike; Greulich, Karl-Otto

    1999-01-01

    Fluorescence in situ hybridization is used to study the arrangement of chromosomes in interphase nuclei of unsynchronized human lymphocytes. DNA probes specific for telomeric DNA, centromeric (alpha) -satellite DNA and whole chromosomes 2, 7, 9 and X are employed. It is demonstrated that the shape of the chromosome territories is variable in cycling cells, for example, close to the metaphase chromosome homologues are arranged pairwise. Furthermore, the relative arrangement of chromosome homologues to each other is not spatially defined. Also, the relative orientation of centromeres and telomeres within a chromosome domain is variable.

  16. DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.

    PubMed

    Vago, Riccardo; Leva, Valentina; Biamonti, Giuseppe; Montecucco, Alessandra

    2009-08-15

    DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.

  17. Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex.

    PubMed

    Ozaki, Shogo; Noguchi, Yasunori; Hayashi, Yasuhisa; Miyazaki, Erika; Katayama, Tsutomu

    2012-10-26

    In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

  18. DNA binding, photo-induced DNA cleavage and cytotoxicity studies of lomefloxacin and its transition metal complexes

    NASA Astrophysics Data System (ADS)

    Ragheb, Mohamed A.; Eldesouki, Mohamed A.; Mohamed, Mervat S.

    2015-03-01

    This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin (LMF) and its ternary transition metal complexes with glycine. The nature of the binding interactions between compounds and calf thymus DNA (CT-DNA) was studied by electronic absorption spectra, fluorescence spectra and thermal denaturation experiments. The obtained results revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode. Furthermore, the DNA cleavage activities of the compounds were investigated by gel electrophoresis. Mechanistic studies of DNA cleavage suggest that singlet oxygen (1O2) is likely to be the cleaving agent via an oxidative pathway, except for Cu(II) complex which proceeds via both oxidative and hydrolytic pathways. Antimicrobial and antitumor activities of the compounds were also studied against some kinds of bacteria, fungi and human cell lines.

  19. Ultrafast Hydration Dynamics Probed by Tryptophan at Protein Surface and Protein-DNA Interface

    NASA Astrophysics Data System (ADS)

    Qin, Yangzhong

    As we all live in a special water planet Earth, the significance of water to life has been universally recognized. The reason why water is so important to life has intrigued many researchers. This dissertation will focus on the ultrafast dynamics of protein surface water and protein-DNA interfacial water which have direct importance to the protein structure and function. Using tryptophan as an intrinsic fluorescence probe, combined with site-directed mutagenesis and ultrafast fluorescence up-conversion spectroscopy, we can achieve single residue spatial resolution and femtosecond temporal resolution. We can also precisely determine the local hydration water dynamics by monitoring the Stokes shift of tryptophan one at a time. Previously, the protein surface hydration has been extensively studied by our group. In this thesis, we will provide more details on the methods we are using to extract the hydration dynamics, and also validate our methods from both experimental and theoretical perspectives. To further interrogate the interfacial water hydration dynamics relative to the protein surface hydration, we studied two DNA polymerases: DNA Polymerase IV (Dpo4) and DNA Polymerase Beta (Pol beta). Both proteins show typical surface hydration pattern with three distinct time components including: (i) the ultrafast sub-picosecond component reflects the bulk type water motion; (ii) a few picoseconds component shows the inner water relaxation mainly corresponding to the local libration and reorientation; (iii) the tens to hundred picoseconds component represents the water-protein coupled motion involving the whole water network reorganization. Dpo4, a loosely DNA binding protein, exhibits very flexible interfacial water which resembles its surface water yet with a significantly reduced ultrafast component. Such dynamic interfacial water not only maintains interfacial flexibility, but also contributes to the low fidelity of the protein. In contrast to the Dpo4, pol beta

  20. Simulation of 125I induced DNA strand breaks in a CAP-DNA complex.

    PubMed

    Li, W; Friedland, W; Jacob, P; Paretzke, H G; Panyutin, I; Neumann, R D

    2002-01-01

    The E. coli catabolite gene activator protein (CAP)-DNA complex with 125I located at the position of the H5 atom of the cytosine near the centre was incorporated into the PARTRAC track structure code. DNA strand breaks due to irradiation were calculated by track structure and radical attack simulations; strand breaks due to neutralisation of the highly charged 125Te ion were derived from a semi-empirical distribution. According to the calculations, the neutralisation effect dominates the strand breakage frequency at 2 bases away from the 125I decay site on both strands. The first breakage distribution counted from a 32P labelled end on the strand with 125I agreed well with experimental data, but on the opposite strand, the calculated distribution is more concentrated around the decay site and its yield is about 20% larger than the measured data.

  1. Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54(nrb)) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

  2. Differential furanose selection in the active sites of archaeal DNA polymerases probed by fixed-conformation nucleotide analogues

    PubMed Central

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L.

    2012-01-01

    DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs. PMID:23050956

  3. DNA interaction studies of a platinum (II) complex containing an antiviral drug, ribavirin: the effect of metal on DNA binding.

    PubMed

    Shahabadi, Nahid; Mirzaei kalar, Zeinab; Moghadam, Neda Hosseinpour

    2012-10-01

    The water-soluble Pt (II) complex, [PtCl (DMSO)(N(4)N(7)-ribavirin)]· H(2)O (ribavirin is an antiviral drug) has been synthesized and characterized by physico-chemical and spectroscopic methods. The binding interactions of this complex with calf thymus DNA (CT-DNA) were investigated using fluorimetry, spectrophotometry, circular dichroism and viscosimetry. The complex binds to CT-DNA in an intercalative mode. The calculated binding constant, K(b), was 7.2×10(5) M(-1). In fluorimetric studies, the enthalpy (ΔH<0) and entropy (ΔS>0) changes of the reaction between the Pt (II) complex with CT-DNA showed hydrophobic interaction. In addition, CD study showed stabilization of the right-handed B form of CT-DNA. All these results prove that the complex interacts with CT-DNA via intercalative mode of binding. In comparison with the previous study of the DNA interaction with ribavirin, these results show that platinum complex has greater affinity to CT-DNA.

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

    SciTech Connect

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

    1987-11-01

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

  5. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-08-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events.

  6. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    PubMed Central

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-01-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events. PMID:27574102

  7. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

  8. Zinc(II)-Coordination Complexes as Membrane Active Fluorescent Probes and Antibiotics

    PubMed Central

    DiVittorio, Kristy M.; Leevy, W. Matthew; O’Neil, Edward J.; Johnson, James R.; Vakulenko, Sergei; Morris, Joshua D.; Rosek, Kristine D.; Serazin, Nathan; Hilkert, Sarah; Hurley, Scott; Marquez, Manuel; Smith, Bradley D.

    2010-01-01

    Molecular probes with zinc(II)-(2,2’-dipicolylamine) coordination complexes associate with oxyanions in aqueous solution and target biomembranes that contain anionic phospholipids. This study examines a new series of coordination complexes with 2,6-bis(zinc(II)-dipicolylamine) phenoxide as the molecular recognition unit. Two lipophilic analogues are observed to partition into the membranes of zwitterionic and anionic vesicles and induce the transport of phospholipids and hydrophilic anions (carboxyfluorescein). These lipophilic zinc complexes are moderately toxic to mammalian cells. A more hydrophilic analogue does not exhibit mammalian cell toxicity (LD50 >50 µg/mL), but it is highly active against the Gram-positive bacteria Staphylococcus aureus (MIC of 1 µg/mL). Furthermore, it is active against clinically important S. aureus strains that are resistant to various antibiotics including vancomycin and oxacillin. The antibiotic action is attributed to its ability to depolarize the bacterial cell membrane. The intense bacterial staining exhibited by a fluorescent conjugate suggests that this family of zinc coordination complexes can be used as molecular probes for the detection and imaging of bacteria. PMID:18076009

  9. Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin

    SciTech Connect

    Pehrson, J.R. )

    1989-12-01

    Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (ii) The DNA in H1-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with H1-stripped chromatin. (iii) The internucleosomal (linker) DNA appears to be relatively straight.

  10. Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay.

    PubMed

    Jung, Cheulhee; Chung, Ji Won; Kim, Un Ok; Kim, Min Hwan; Park, Hyun Gyu

    2011-01-15

    We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.

  11. Spectroscopic studies on the interaction of quercetin-terbium(III) complex with calf thymus DNA.

    PubMed

    Dehghan, Gholamreza; Dolatabadi, Jafar Ezzati Nazhad; Jouyban, Abolghasem; Zeynali, Karim Asadpour; Ahmadi, Seyed Mojtaba; Kashanian, Soheila

    2011-03-01

    The interaction of native calf thymus DNA (CT-DNA) with quercetin-terbium(III) [Q-Tb(III)] complex at physiological pH was monitored by UV absorption spectrophotometry, circular dichroism, fluorescence spectroscopy, and viscosimetric techniques. The complex displays binding properties to the CT-DNA and was found to interact with CT-DNA through outside binding, demonstrated by a hypochromic effect of Q-Tb(III) on the UV spectra of CT-DNA and the calculated association constants (K). Also, decrease in the specific viscosity of CT-DNA, decrease in the fluorescence intensity of Q-Tb(III) solutions in the presence of increasing amounts of CT-DNA, and detectable changes in the circular dichroism spectrum of CT-DNA are other evidences to indicate that Q-Tb(III) complex interact with CT-DNA through outside binding.

  12. Surface modification of poly(dimethylsiloxane) (PDMS) microchannels with DNA capture-probes for potential use in microfluidic DNA analysis systems

    NASA Astrophysics Data System (ADS)

    Khodakov, Dmitriy A.; Thredgold, Leigh D.; Lenehan, Claire E.; Andersson, Gunther A.; Kobus, Hilton; Ellis, Amanda V.

    2011-12-01

    Poly(dimethylsiloxane) (PDMS) is an elastomeric material used for microfluidic devices and is especially suited to medical and forensic applications. This is due to its relatively low cost, ease of fabrication, excellent optical transmission characteristics and its ability to support electroosmotic flow, required during electrophoretic separations. These aspects combined with its large range of surface modification chemistries, make PDMS an attractive substrate in microfluidic devices for, in particular, DNA separation. Here, we report the successful wet chemical surface modification of PDMS microchannels using a simple three step method to produce an isothiocyanate-terminated surface. Initially, PDMS was oxygen plasma treated to produce a silanol-terminated surface, this was then reacted with 3-aminopropyltriethoxysilane with subsequent reaction of the now amine-terminated surface with p-phenylenediisothiocyanate. Water contact angle measurements both before and after modification showed a reduction in hydrophobicity from 101o for native PDMS to 94o for the isothiocyante-terminated PDMS. The isothiocyanate-terminated surface was then coupled with an amineterminated single-stranded DNA (ssDNA) oligonucleotide capture probe via a thiourea linkage. Confirmation of capture probe attachment was observed using fluorescent microscopy after hybridization of the capture probes with fluorescently labeled complimentary ssDNA oligonucleotides.

  13. Ca K-Edge XAS as a Probe of Calcium Centers in Complex Systems

    PubMed Central

    2014-01-01

    Herein, Ca K-edge X-ray absorption spectroscopy (XAS) is developed as a means to characterize the local environment of calcium centers. The spectra for six, seven, and eight coordinate inorganic and molecular calcium complexes were analyzed and determined to be primarily influenced by the coordination environment and site symmetry at the calcium center. The experimental results are closely correlated to time-dependent density functional theory (TD-DFT) calculations of the XAS spectra. The applicability of this methodology to complex systems was investigated using structural mimics of the oxygen-evolving complex (OEC) of PSII. It was found that Ca K-edge XAS is a sensitive probe for structural changes occurring in the cubane heterometallic cluster due to Mn oxidation. Future applications to the OEC are discussed. PMID:25492398

  14. Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA

    SciTech Connect

    Sharma A.; Heroux A.; Jenkins K. R.; Bowman G. D.

    2011-12-09

    Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves.

  15. Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA.

    PubMed

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-11-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

  16. Sequential picosecond isomerizations in a photochromic ruthenium sulfoxide complex triggered by pump-repump-probe spectroscopy.

    PubMed

    King, Albert W; Jin, Yuhuan; Engle, James T; Ziegler, Christopher J; Rack, Jeffrey J

    2013-02-18

    The complex [Ru(bpy)(2)(bpSO)](PF(6))(2), where bpy is 2,2'-bipydine and bpSO is 1,2-bis(phenylsulfinyl)ethane, exhibits three distinct isomers which are accessible upon metal-to-ligand charge-transfer (MLCT) irradiation. This complex and its parent, [Ru(bpy)(2)(bpte)](PF(6))(2), where bpte is 1,2-bis(phenylthio)ethane, have been synthesized and characterized by UV-visible spectroscopy, NMR, X-ray crystallography, and femtosecond transient absorption spectroscopy. A novel method of 2-color Pump-Repump-Probe spectroscopy has been employed to investigate all three isomers of the bis-sulfoxide complex. This method allows for observation of the isomerization dynamics of sequential isomerizations of each sulfoxide from MLCT irradiation of the S,S-bonded complex to ultimately form the O,O-bonded metastable complex. One-dimensional (1-D) and two-dimensional (2-D) (COSY, NOESY, and TOCSY) (1)H NMR data show the thioether and ground state S,S-bonded sulfoxide complexes to be rigorously C(2) symmetric and are consistent with the crystal structures. Transient absorption spectroscopy reveals that the S,S to S,O isomerization occurs with an observed time constant of 56.8 (±7.4) ps. The S,O to O,O isomerization time constant was found to be 59 (±4) ps by pump-repump-probe spectroscopy. The composite S,S- to O,O-isomer quantum yield is 0.42.

  17. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    PubMed Central

    Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

    2009-01-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

  18. Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes.

    PubMed

    Valdez-Moreno, M; Ivanova, N V; Elías-Gutiérrez, M; Contreras-Balderas, S; Hebert, P D N

    2009-02-01

    The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0.45%, while congeneric taxa showed 5.1% divergence. Three species of Poblana, each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (<2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax-Astyanax complex. The symbranchid, Opisthernon aenigmaticum, has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.

  19. Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair*

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Pestryakov, Pavel E.; Petruseva, Irina O.; Sugasawa, Kaoru; Chen, Xuejing; Min, Jung-Hyun; Lavrik, Olga I.

    2013-01-01

    The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (KD ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nm, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms. PMID:23443653

  20. Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe.

    PubMed

    Zhang, Hongge; Wang, Minjuan; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2011-05-15

    A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F(0)=2.73 C (μM)+1.14 (R=0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N=3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.

  1. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.

    PubMed

    D'Amours, Damien; Jackson, Stephen P

    2002-05-01

    The Mre11 complex is a multisubunit nuclease that is composed of Mre11, Rad50 and Nbs1/Xrs2. Mutations in the genes that encode components of this complex result in DNA- damage sensitivity, genomic instability, telomere shortening and aberrant meiosis. The molecular defect that underlies these phenotypes has long been thought to be related to a DNA repair deficiency. However, recent studies have uncovered functions for the Mre11 complex in checkpoint signalling and DNA replication.

  2. Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing.

    PubMed

    Pumphrey, Graham M; Ranchou-Peyruse, Anthony; Spain, Jim C

    2011-07-01

    Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

  3. A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data

    PubMed Central

    Teschendorff, Andrew E.; Marabita, Francesco; Lechner, Matthias; Bartlett, Thomas; Tegner, Jesper; Gomez-Cabrero, David; Beck, Stephan

    2013-01-01

    Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely different DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normalization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a statistical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favourably with two competing methods. Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform. Availability: BMIQ is freely available from http://code.google.com/p/bmiq/. Contact: a.teschendorff@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online PMID:23175756

  4. Analytical methods to determine the comparative DNA binding studies of curcumin-Cu(II) complexes.

    PubMed

    Rajesh, Jegathalaprathaban; Rajasekaran, Marichamy; Rajagopal, Gurusamy; Athappan, Periakaruppan

    2012-11-01

    DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of K(b), 5.21×10(4)M(-1) that are higher than that obtained for 2 (red-shift, 2 nm; K(b), 1.73×10(4)M(-1)) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the E(pc) and E(0)' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HO()) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

  5. Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

    PubMed

    Jones, Maiysha D; Singleton, David R; Sun, Wei; Aitken, Michael D

    2011-05-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

  6. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil▿†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the