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Sample records for dna duplexes modified

  1. Evaluation of ion binding to DNA duplexes using a size-modified Poisson-Boltzmann theory.

    Science.gov (United States)

    Chu, Vincent B; Bai, Yu; Lipfert, Jan; Herschlag, Daniel; Doniach, Sebastian

    2007-11-01

    Poisson-Boltzmann (PB) theory is among the most widely applied electrostatic theories in biological and chemical science. Despite its reasonable success in explaining a wide variety of phenomena, it fails to incorporate two basic physical effects, ion size and ion-ion correlations, into its theoretical treatment. Recent experimental work has shown significant deviations from PB theory in competitive monovalent and divalent ion binding to a DNA duplex. The experimental data for monovalent binding are consistent with a hypothesis that attributes these deviations to counterion size. To model the observed differences, we have generalized an existing size-modified Poisson-Boltzmann (SMPB) theory and developed a new numerical implementation that solves the generalized theory around complex, atomistic representations of biological molecules. The results of our analysis show that good agreement to data at monovalent ion concentrations up to approximately 150 mM can be attained by adjusting the ion-size parameters in the new size-modified theory. SMPB calculations employing calibrated ion-size parameters predict experimental observations for other nucleic acid structures and salt conditions, demonstrating that the theory is predictive. We are, however, unable to model the observed deviations in the divalent competition data with a theory that only accounts for size but neglects ion-ion correlations, highlighting the need for theoretical descriptions that further incorporate ion-ion correlations. The accompanying numerical solver has been released publicly, providing the general scientific community the ability to compute SMPB solutions around a variety of different biological structures with only modest computational resources.

  2. Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

    Energy Technology Data Exchange (ETDEWEB)

    Diop-Frimpong, Benjamin; Prakash, Thazha P.; Rajeev, Kallanthottathil G.; Manoharan, Muthiah; Egli, Martin (Isis Pharm.); (Alnylam Pharm.); (Vanderbilt)

    2010-03-05

    Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4'-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4'-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s{sup 2}U) and 5-methyl-2-thiouridine (m{sup 5}s{sup 2}U) are natural nucleotide analogs. s{sup 2}U in tRNA confers greater specificity of codon-anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2'-deoxyribose sugars for a C3'-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2'-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2'-O-[2-(methoxy)ethyl]-2-thiothymidine (m{sup 5}s{sup 2}Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m{sup 5}s{sup 2}Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m{sup 5}Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter 'opening', an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m{sup 5}s{sup 2}Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of

  3. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...... protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form...

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

    Science.gov (United States)

    Wong, Jiun Ru; Shao, Fangwei

    2017-01-01

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

  5. Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity.

    Science.gov (United States)

    Lee, Chang Yeol; Park, Ki Soo; Park, Hyun Gyu

    2017-12-15

    We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Thermodynamic and kinetic characterization of duplex formation between 2'-O, 4'-C-methylene-modified oligoribonucleotides, DNA and RNA

    DEFF Research Database (Denmark)

    Christensen, Ulla

    2007-01-01

    2'-O,4'-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid...

  7. Energetics, conformation, and recognition of DNA duplexes modified by monodentate Ru(II) complexes containing terphenyl arenes

    Czech Academy of Sciences Publication Activity Database

    Nováková, Olga; Malina, Jaroslav; Suchánková, T.; Kašpárková, Jana; Bugarcic, T.; Sadler, P.J.; Brabec, Viktor

    2010-01-01

    Roč. 16, č. 19 (2010), s. 5744-5754 ISSN 0947-6539 R&D Projects: GA ČR(CZ) GD301/09/H004; GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) ME10066; GA MŠk(CZ) OC08003; GA AV ČR(CZ) KAN200200651 Grant - others:GA MŠk(CZ) OC09018 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : calorimetry * ruthenium * DNA recognition Subject RIV: BO - Biophysics Impact factor: 5.476, year: 2010

  8. Spermine Condenses DNA, but Not RNA Duplexes

    Energy Technology Data Exchange (ETDEWEB)

    Katz, Andrea M.; Tolokh, Igor S.; Pabit, Suzette A.; Baker, Nathan; Onufriev, Alexey V.; Pollack, Lois

    2017-01-01

    Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA, and some RNAs such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA, but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA and compare our findings with predictions of molecular dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence, containing a mixture of 14 GC pairs and 11 AU pairs, resists condensation relative to DNA of an equivalent sequence or to 25 base pair poly(rA):poly(rU) RNA. Comparison of wide-angle x-ray scattering profiles with simulation suggests that spermine is sequestered deep within the major groove of mixed sequence RNA, preventing condensation by limiting opportunities to bridge to other molecules as well as stabilizing the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds external to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble, and available for interaction with other molecules in the cell, despite the presence of spermine at concentrations high enough to precipitate DNA.

  9. Detection method for genetically modified papaya using duplex PCR.

    Science.gov (United States)

    Yamaguchi, Akihiro; Shimizu, Kaori; Mishima, Takashi; Aoki, Nobutaro; Hattori, Hideki; Sato, Hidetaka; Ueda, Nobuo; Watanabe, Takahiro; Hino, Akihiro; Akiyama, Hiroshi; Maitani, Tamio

    2006-08-01

    A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.

  10. The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

    Science.gov (United States)

    Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

    2016-07-01

    Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

  11. Conserved guanine-guanine stacking in tetraplex and duplex DNA

    Czech Academy of Sciences Publication Activity Database

    Kypr, Jaroslav; Fialová, Markéta; Chládková, Jana; Tůmová, Marcela; Vorlíčková, Michaela

    2001-01-01

    Roč. 30, č. 7 (2001), s. 555-558 ISSN 0175-7571 R&D Projects: GA AV ČR IAA5004802 Institutional research plan: CEZ:AV0Z5004920 Keywords : tetraplex DNA * duplex DNA * circular dichroism spectra Subject RIV: BO - Biophysics Impact factor: 1.917, year: 2001

  12. Micromechanics of base pair unzipping in the DNA duplex

    International Nuclear Information System (INIS)

    Volkov, Sergey N; Paramonova, Ekaterina V; Yakubovich, Alexander V; Solov’yov, Andrey V

    2012-01-01

    All-atom molecular dynamics (MD) simulations of DNA duplex unzipping in a water environment were performed. The investigated DNA double helix consists of a Drew-Dickerson dodecamer sequence and a hairpin (AAG) attached to the end of the double-helix chain. The considered system is used to examine the process of DNA strand separation under the action of an external force. This process occurs in vivo and now is being intensively investigated in experiments with single molecules. The DNA dodecamer duplex is consequently unzipped pair by pair by means of the steered MD. The unzipping trajectories turn out to be similar for the duplex parts with G⋅C content and rather distinct for the parts with A⋅T content. It is shown that during the unzipping each pair experiences two types of motion: relatively quick rotation together with all the duplex and slower motion in the frame of the unzipping fork. In the course of opening, the complementary pair passes through several distinct states: (i) the closed state in the double helix, (ii) the metastable preopened state in the unzipping fork and (iii) the unbound state. The performed simulations show that water molecules participate in the stabilization of the metastable states of the preopened base pairs in the DNA unzipping fork. (paper)

  13. 5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

    Science.gov (United States)

    Frank, Natia L.; Meade, Thomas J.

    2003-01-01

    Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

  14. NMR analysis of duplex d(CGCGATCGCG)2 modified by lambda- and delta-[Ru(bpy)2(m-GHK)]Cl2 and DNA photocleavage study

    Czech Academy of Sciences Publication Activity Database

    Myari, A.; Hadjiliadis, N.; Garoufis, A.; Malina, Jaroslav; Brabec, Viktor

    2007-01-01

    Roč. 12, č. 3 (2007), s. 279-292 ISSN 0949-8257 R&D Projects: GA ČR(CZ) GA305/05/2030; GA AV ČR(CZ) KJB400040601; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA MŠk(CZ) LC06030; GA MŠk(CZ) 1P05OC072 Institutional research plan: CEZ:AV0Z50040702 Keywords : Ru(II) * peptide * DNA photocleavage Subject RIV: BO - Biophysics Impact factor: 3.325, year: 2007

  15. Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

    Directory of Open Access Journals (Sweden)

    Fabian Haß

    2017-10-01

    Full Text Available The human-specific Alu elements, belonging to the class of Short INterspersed Elements (SINEs, have been shown to be a powerful tool for population genetic studies. An earlier study in this department showed that it was possible to analyze Alu presence/absence in 3000-year-old skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. We developed duplex Alu screening PCRs with flanking primers for two Alu elements, each combined with a single internal Alu primer. By adding an internal primer, the approximately 400–500 bp presence signals of Alu elements can be detected within a range of less than 200 bp. Thus, our PCR approach is suited for highly fragmented ancient DNA samples, whereas NGS analyses frequently are unable to handle repetitive elements. With this analysis system, we examined remains of 12 individuals from the Lichtenstein cave with different degrees of DNA degradation. The duplex PCRs showed fully informative amplification results for all of the chosen Alu loci in eight of the 12 samples. Our analysis system showed that Alu presence/absence analysis is possible in samples with different degrees of DNA degradation and it reduces the amount of valuable skeletal material needed by a factor of four, as compared with a singleplex approach.

  16. Highly selective DNA biosensor based on the long-range electron transfer of indigo carmine through DNA duplex

    International Nuclear Information System (INIS)

    Wang, Q.; Ding, Y.; Wang, L.; Gao, C.; Gao, F.; Gao, F.

    2012-01-01

    We have developed a highly selective DNA biosensor. It was based on the long-range electron transfer (LRET) from the electroactive dye indigo carmine (IC) through the DNA duplex on a glassy carbon electrode. Voltammetric experiments showed that IC interacts with dsDNA through a typical intercalative mode with a relatively strong affinity of 2.3(±0.6) x 10 6 M -1 . If incubated with DNA in IC solution, no response was observed with the ssDNA-modified probe electrode. However, a pair of well-defined redox peak was observed with a DNA-hybridized electrode, proving the presence of LRET on the biosensor. The biosensor also can differentiate complementary sequences, non-complementary sequences, and even the mutated sequences with single-base mismatches at different sites. (author)

  17. Properties of 5- and/or 2-modified 2'-O-cyanoethyl uridine residue: 2'-O-cyanoethyl-5-propynyl-2-thiouridine as an efficient duplex stabilizing component.

    Science.gov (United States)

    Masaki, Yoshiaki; Miyasaka, Ryuta; Hirai, Kunihiro; Kanamori, Takashi; Tsunoda, Hirosuke; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2014-02-21

    We systematically synthesized eight types of 5- and/or 2-modified uridine derivatives and evaluated their effect on duplex stability. The incorporation of 2'-O-cyanoethyl-2-thio-5-propynyluridine (p(5)s(2)UOCE) into RNA was significantly effective for stabilization of RNA/RNA (+8.5 °C) and DNA/RNA (+10.4 °C) duplexes. These striking effects were maintained in oligonucleotides with different sequences or multiple incorporations. In addition, p(5)s(2)UOCE increased selectivity toward the correct AU Watson-Crick base pair over the most stable mismatched base pair in both RNA/RNA and DNA/RNA duplexes. Hence, p(5)s(2)UOCE could be useful for various applications of modified oligonucleotides that need high duplex stability and base pairing selectivity.

  18. Electrochemical single-molecule conductivity of duplex and quadruplex DNA

    DEFF Research Database (Denmark)

    Zhang, Ling; Zhang, Jingdong; Ulstrup, Jens

    2017-01-01

    Photoinduced and electrochemical charge transport in DNA (oligonucleotides, OGNs) and the notions “hopping”, superexchange, polaron, and vibrationally gated charge transport have been in focus over more than two decades. In recent years mapping of electrochemical charge transport of pure and redox...... marked single- and double-strand OGNs has reached the single-molecule level based i.a. on electrochemical in situ scanning tunnelling microscopy (STM) and break-junction (B-J) STM. There are much fewer such reports on “non-canonical” OGN structures such as G-quadruplexes. We discuss first single......-molecule electrochemical conductivity of pure and redox marked duplex OGNs, and address next electrochemistry and electrochemical conductivity in the few reported monolayer and single-molecule G-quadruplex studies. Facile electrochemical electron transfer of iron protoporphyrin IX stacked onto three-quartet 12-guanine...

  19. Enhanced H-bonding and pi-stacking in DNA: a potent duplex-stabilizing and mismatch sensing nucleobase analogue

    DEFF Research Database (Denmark)

    Lou, Chenguang; Dallmann, Andre; Marafini, Pietro

    2014-01-01

    X-pyrene is a new nucleic acid duplex stabilizing cytosine analogue that combines enhanced pi-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrids. X-pyrene is highly selective for guanine as a partner and duplex...

  20. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Directory of Open Access Journals (Sweden)

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  1. Different models for phosphate anion shielding in DNA duplexes: An elaboration with ab initio and molecular mechanics calculations

    Science.gov (United States)

    Buck, Henk

    In an effort to overcome a significant difference between high-level ab initio calculations and X-ray data of DNA duplexes, Fonseca Guerra et al. 9-11 studied a number of model systems for A-T and G-C basepairs at various levels of nonlocal Density Functional Theory. There was an excellent agreement with the gas-phase experimental bond enthalpies for the A-T and G-C basepairs. On the other hand the hydrogen bond lengths between the bases differ from the X-ray results. After introduction of a molecular environment as local water and Na+ ions, the agreement between theory and experiment was excellent. However, careful analysis shows that this picture is far from correct. In fact, the model was constructed as a backbone-modified DNA duplex in which the nonbonding oxygens of the phosphate linkages are completely shielded by proton addition. Experimental results with respect to backbone-modified DNAs clearly show that changes in the backbone focused on phosphate shielding result in DNA duplexes with a variety in conformational behavior. In addition to an analysis of the aforementioned contradiction, we also give molecular mechanics calculations which show that the A-T and G-C bond enthalpies are of the same order as the corresponding results of Fonseca Guerra et al. under the condition of complete anionic shielding of the nonbonded oxygens in the phosphate linkages.

  2. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Science.gov (United States)

    Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  3. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  4. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

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

    DEFF Research Database (Denmark)

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

    2011-01-01

    We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization...... to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple...... sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both...

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

    Science.gov (United States)

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

    2016-05-19

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

  7. NMR studies of abasic sites in DNA duplexes: Deoxyadenosine stacks into the helix opposite acyclic lesions

    International Nuclear Information System (INIS)

    Kalnik, M.W.; Chang, Chienneng; Johnson, F.; Grollman, A.P.; Patel, D.J.

    1989-01-01

    Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C)·d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated AP P 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C-)·d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated AP E 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the AP P 9-mer and AP E 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H 2 O and D 2 O solution between -5 and 5 degree C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and El14) and stacks between the flanking G4·C15 and G6·C13 Watson-Crick base pairs in both the AP P 9-mer and AP E 9-mer duplexes. Proton NMR parameters for the Ap P 9-mer and AP E 9-mer duplexes are similar to those reported previously. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the AP P 9-mer and AP E 9-mer duplexes (5 degree C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles

  8. Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry

    Science.gov (United States)

    Griffey, Richard H.; Greig, Michael J.

    1997-05-01

    The identify and location of base pair mismatches in non- covalent DNA:RNA duplexes are established using MS and MS-MS on a quadruple ion trap with electrospray ionization (ESI). MS-MS experiments on a 14mer duplex (D) with a single C:A base pair mismatch using lower activation energy results in selective cleavage of the mismatched A nucleobase, even in the presence of the wild-type duplex. The location of the mismatch base pair can be discerned via presence of the wild-type duplex. The location of the mismatch base pair can be discerned via selection of the (D-5H)5- ion and fragmentation of the backbone at that location in a n additional MS-MS experiment. Selective fragmentation is observed for C in a C-C mismatched base pair, which is very difficult to detect using chemical cleavage or E. coli mismatch binding protein. In an RNA:DNA duplex with a single base pair mismatch, the DNA base is removed without fragmentation of the RNA strand, greatly simplifying the interpretation of the resulting MS spectrum. A method is presented for detecting two DNA strands, for example a point mutation which generates an oncogenic phenotype, and the wild-type message. The results suggest that ESI-MS-MS may provide a rapid and selective method to identify and locate genetic mutations without the need for chemical degradation or protein binding followed by gel electrophoresis.

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

    Science.gov (United States)

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

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

  10. Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G x T and G x O4meT pairing in dodecanucleotide duplexes

    International Nuclear Information System (INIS)

    Kalnik, M.W.; Kouchakdjian, M.; Li, B.F.L.; Swann, P.F.; Patel, D.J.

    1988-01-01

    High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G x T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O 4 meT-G-C-G) duplex (designated G x O 4 meT 12-mer) containing G x T and G x O 4 meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G x T 12-mer and G x O 4 meT 12-mer duplexes in H 2 O and D 2 O solution. The guanosine and thymidine imino protons in the G x T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G x T 12-mer duplex. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G x T mismatch or in G x C base pairs indicates that hydrogen bonding to O 4 meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH 3 group of O 4 meT across the pair and NOEs to the imino protons of flanking base pairs. Taken together with data from the NMR of nonexchangeable protons, this shows that both G and O 4 meT have anti-glycosidic torsion angles and are stacked into the duplex. Comparison of the intensity of the NOEs between the guanosine imino proton and the OCH 3 of O 4 meT as well as other protons in its vicinity demonstrates that the OCH 3 group of O 4 meT adopts the syn orientation with respect to N3 of the methylated thymidine. The authors propose an alternate base pairing mode stabilized by one short hydrogen bond between the 2-amino group of guanosine and the 2-carbonyl group of O 4 met

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

    NARCIS (Netherlands)

    Pohjoismaki, J.L.; Holmes, J.B.; Wood, S.R.; Yang, M.Y.; Yasukawa, T.; Reyes, A.; Bailey, L.J.; Cluett, T.J.; Goffart, S.; Willcox, S.; Rigby, R.E.; Jackson, A.P.; Spelbrink, J.N.; Griffith, J.D.; Crouch, R.J.; Jacobs, H.T.; Holt, I.J.

    2010-01-01

    We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of

  12. New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.

    Science.gov (United States)

    Zhou, Huiqing; Hintze, Bradley J; Kimsey, Isaac J; Sathyamoorthy, Bharathwaj; Yang, Shan; Richardson, Jane S; Al-Hashimi, Hashim M

    2015-04-20

    Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson-Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1'-C1' distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1'-C1' distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5'-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2'-alkylated RNA monomers.

    Science.gov (United States)

    Karmakar, Saswata; Madsen, Andreas S; Guenther, Dale C; Gibbons, Bradley C; Hrdlicka, Patrick J

    2014-10-21

    Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and--more recently--engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.

  14. Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

    DEFF Research Database (Denmark)

    Izvolsky, K I; Demidov, V V; Nielsen, P E

    2000-01-01

    I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

  15. Comparison of positively charged DNG with DNA duplexes: a computational approach.

    Science.gov (United States)

    Toporowski, Joseph W; Reddy, Swarnalatha Y; Bruice, Thomas C

    2005-06-01

    Molecular dynamics is used to investigate the structural properties of the cationic DNA analogue deoxynucleic guanidine (DNG), in which a guanidinium group replaces the phosphate moiety of DNA. This study examines the DNG duplex dodecamers d(Ag)(12).d(Tg)(12) and d(Gg)(12).d(Cg)(12), as well as their DNA counterparts. Watson-Crick base-pairing is maintained in the solvated DNG duplex models during the 5ns simulations. The idealized DNG dodecamers assume many parameters characteristic of the corresponding native DNA, assuming B-DNA conformations. Several helical parameters are rather unique to DNG, including buckle, slide, inclination, propeller, and X-displacement. Fewer transitions in backbone torsions occur in the DNG duplexes compared to those of the DNA, which may result from the greater rigidity of the sp(2) hybridized guanidinium group verses the flexible sp(3) phosphate group. The DNG helices have exceptionally shallow major grooves and very deep minor grooves. The major and minor groove widths of DNG are narrower than those of the respective DNA counterparts.

  16. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    Science.gov (United States)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  17. Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

    OpenAIRE

    Bergeron, François; Houde, Daniel; Hunting, Darel J.; Wagner, J. Richard

    2004-01-01

    2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences ...

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

    Science.gov (United States)

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

    2015-07-01

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

  19. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

    Science.gov (United States)

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-05-23

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

  20. Impact of bistrand abasic sites and proximate orientation on DNA global structure and duplex energetics.

    Science.gov (United States)

    Minetti, Conceição A; Sun, Jeffrey Y; Jacobs, Daniel P; Kang, Inkoo; Remeta, David P; Breslauer, Kenneth J

    2018-01-11

    Bistrand lesions embedded within a single helical turn of tridecameric deoxyoligonucleotide duplexes represent a model system for exploring the impact of clustered lesions that occur in vivo and pose a significant challenge to cellular repair machineries. Such investigations are essential for understanding the forces that dictate lesion-induced mutagenesis, carcinogenesis, and cytotoxicity within a context that mimics local helical perturbations caused by an ionizing radiation event. This study characterizes the structural and energy profiles of DNA duplexes harboring synthetic abasic sites (tetrahydrofuran, F) as models of clustered bistrand abasic (AP) lesions. The standard tridecameric dGCGTACCCATGCG·dCGCATGGGTACGC duplex is employed to investigate the energetic impact of single and bistrand AP sites by strategically replacing one or two bases within the central CCC/GGG triplet. Our combined analysis of temperature-dependent UV and circular dichroism (CD) profiles reveals that the proximity and relative orientation of AP sites within bistrand-damaged duplexes imparts a significant thermodynamic impact. Specifically, 3'-staggered lesions (CCF/GFG) exert a greater destabilizing effect when compared with their 5'-counterpart (FCC/GFG). Moreover, a duplex harboring the central bistrand AP lesion (CFC/GFG) is moderately destabilized yet exhibits distinct properties relative to both the 3' and 5'-orientations. Collectively, our energetic data are consistent with structural studies on bistrand AP-duplexes of similar sequence in which a 3'-staggered lesion exerts the greatest perturbation, a finding that provides significant insight regarding the impact of orientation on lesion repair processing efficiency. © 2018 The Authors Biopolymers Published by Wiley Periodicals, Inc.

  1. Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics

    Science.gov (United States)

    Holmstrom, Erik D.; Dupuis, Nicholas F.; Nesbitt, David J.

    2014-01-01

    Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10−11 liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20–100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation. PMID:24411254

  2. Carotid Consensus Panel duplex criteria can replace modified University of Washington criteria without affecting accuracy.

    Science.gov (United States)

    Kim, Ann H; Augustin, Gener; Shevitz, Andrew; Kim, Hannah; Trivonovich, Michael R; Powell, Alexis R; Kumins, Norman; Tarr, Robert; Kashyap, Vikram S

    2018-03-01

    The decision to intervene for internal carotid stenosis often depends on the degree of stenosis seen on duplex ultrasound (US). The aim of this study is to compare the diagnostic accuracy of two criteria: modified University of Washington (UW) and 2003 Carotid Consensus Panel (CCP). All patients undergoing US in an accredited (IAC) vascular laboratory from January 2010 to June 2015 were reviewed ( n=18,772 US exams). Patients receiving a neck computed tomography angiography (CTA) within 6 months of the US were included in the study ( n=254). The degree of stenosis was determined by UW/CCP criteria and confirmed on CTA images using North American Symptomatic Carotid Endarterectomy Trial (NASCET)/European Carotid Surgery Trial (ECST) schema. Kappa analysis with 95% confidence intervals (CIs) were utilized to determine duplex-CTA agreement. A total of 417 carotid arteries from 221 patients were assessed in this study. The modified UW criteria accurately classified 266 (63.9%, kappa = 0.321, 95% CI 0.255 to 0.386) cases according to NASCET-derived measurements. The sensitivity, specificity, and accuracy at ≥ 60% stenosis were 65.7%, 81.3%, and 81.9%. The CCP criteria resulted in 296 (70.9%) accurate diagnoses (kappa = 0.359, 95% CI 0.280 to 0.437). At ≥ 70% stenosis, the sensitivity, specificity and accuracy were 38.8%, 91.6%, and 87.1% for NASCET. Comparison of the duplex results to ECST-derived CTA measurements revealed a similar trend (UW 53.1%, κ = 0.301 vs CCP 62.1%, κ = 0.315). The CCP criteria demonstrate a higher concordance rate with measurements taken from CTAs. The CCP criteria may be more sensitive in classifying clinically significant degrees of stenosis without a loss in diagnostic accuracy.

  3. Correlation between the thermodynamic stability of DNA duplexes and the interaction and solvation energies of DNA building blocks

    Czech Academy of Sciences Publication Activity Database

    Řezáč, Jan; Hobza, Pavel

    2008-01-01

    Roč. 73, č. 2 (2008), s. 161-174 ISSN 0010-0765 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0009; GA ČR(CZ) GD203/05/H001 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA duplex * interaction energy * stability Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.784, year: 2008

  4. QCM-based rupture force measurement as a tool to study DNA dehybridization and duplex stability.

    Science.gov (United States)

    Dultsev, Fedor N; Kolosovsky, Eugeny A; Lomzov, Alexander A; Pyshnyi, Dmitrii V

    2017-02-01

    The stability of double-stranded DNA (dsDNA) was assessed on the basis of unwinding force measurement. Unwinding force was measured directly with a quartz crystal microbalance (QCM). The amplitude of its surface oscillations was controlled by supplying variable alternate voltage. Under smoothly increasing amplitude of QCM surface oscillations, dsDNA fixed on QCM surface through one of its ends got unwound. This procedure allows reliable measurement of rupture force as small as 5-10 pN. It was demonstrated that oscillations of the surface, with dsDNA bound through one of its ends to this surface, at a frequency of 14 MHz, cause helix unwinding to form two complementary parts due to viscous forces of the liquid medium. Unwinding starts at the upper end. This was proven using oligonucleotide duplexes containing mismatches in different positions. For duplexes containing complementary 20 base pairs, the helix unwinding force is equal to 30-40 pN, which is in agreement with the data obtained by means of atomic-force microscopy (AFM) for the case of unzipping mode. Graphical Abstract Rupture force depending on mismatch position in dsDNA.

  5. Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers†

    Science.gov (United States)

    Karmakar, Saswata; Madsen, Andreas S.; Guenther, Dale C.; Gibbons, Bradley C.; Hrdlicka, Patrick J.

    2014-01-01

    Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔTm/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics. PMID:25144705

  6. DNA polymerase having modified nucleotide binding site for DNA sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  7. Studies of DNA dumbbells VII: evaluation of the next-nearest-neighbor sequence-dependent interactions in duplex DNA.

    Science.gov (United States)

    Owczarzy, R; Vallone, P M; Goldstein, R F; Benight, A S

    1999-01-01

    Melting experiments were conducted on 22 DNA dumbbells as a function of solvent ionic strength from 25-115 mM Na(+). The dumbbell molecules have short duplex regions comprised of 16-20 base pairs linked on both ends by T(4) single-strand loops. Only the 4-8 central base pairs of the dumbbell stems differ for different molecules, and the six base pairs on both sides of the central sequence and adjoining loops on both ends are the same in every molecule. Results of melting analysis on the 22 new DNA dumbbells are combined with our previous results on 17 other DNA dumbbells, with stem lengths containing from 14-18 base pairs, reported in the first article of this series (Doktycz, Goldstein, Paner, Gallo, and Benight, Biopoly 32, 1992, 849-864). The combination of results comprises a database of optical melting parameters for 39 DNA dumbbells in ionic strengths from 25-115 mM Na(+). This database is employed to evaluate the thermodynamics of singlet, doublet, and triplet sequence-dependent interactions in duplex DNA. Analysis of the 25 mM Na(+) data reveals the existence of significant sequence-dependent triplet or next-nearest-neighbor interactions. The enthalpy of these interactions is evaluated for all possible triplets. Some of the triplet enthalpy values are less than the uncertainty in their evaluation, indicating no measurable interaction for that particular sequence. This finding suggests that the thermodynamic stability of duplex DNA depends on solvent ionic strength in a sequence-dependent manner. As a part of the analysis, the nearest-neighbor (base pair doublet) interactions in 55, 85, and 115 mM Na(+) are also reevaluated from the larger database. Copyright 2000 John Wiley & Sons, Inc.

  8. Unstable Hoogsteen base pairs adjacent to echinomycin binding sites within a DNA duplex

    International Nuclear Information System (INIS)

    Gilbert, D.E.; van der Marel, G.A.; van Boom, J.H.; Feigon, J.

    1989-01-01

    The bisintercalation complex present between the DNA octamer [d(ACGTACGT)] 2 and the cyclic octadepsipeptide antibiotic echinomycin has been studied by one- and two-dimensional proton NMR, and the results obtained have been compared with the crystal structures of related DNA-echinomycin complexes. Two echinomycins are found to bind cooperatively to each DNA duplex at the CpG steps, with the two quinoxaline rings of each echinomycin bisintercalating between the C·G and A·T base pairs. At low temperatures, the A·T base pairs on either side of the intercalation site adopt the Hoogsteen conformation, as observed in the crystal structures. However, as the temperature is raised, the Hoogsteen base pairs in the interior of the duplex are destabilized and are observed to be exchanging between the Hoogsteen base pair and either an open or a Watson-Crick base-paired state. The terminal A·T base pairs, which are not as constrained by the helix as the internal base pairs, remain stably Hoogsteen base-paired up to at least 45 degree C. The implications of these results for the biological role of Hoogsteen base pairs in echinomycin-DNA complexes in vivo are discussed

  9. Combined Triplex/Duplex Invasion of Double-Stranded DNA by "Tail-Clamp" Peptide Nucleic Acid

    DEFF Research Database (Denmark)

    Bentin, Thomas; Larsen, H. J.; Nielsen, Peter E.

    2003-01-01

    "Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension...... as determined by T-m measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C-50 measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed...... to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology....

  10. Optimizing anti-gene oligonucleotide 'Zorro-LNA' for improved strand invasion into duplex DNA

    DEFF Research Database (Denmark)

    Zaghloul, Eman M; Madsen, Andreas S; Moreno, Pedro M D

    2011-01-01

    established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3'-5'-5'-3' single-stranded Zorro-LNA (ssZorro) by using both 3'- and 5'-phosphoramidites. With ssZorro, a significantly greater extent......Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now...... and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested...

  11. Interaction of anticancer Ru(III) complexes with single stranded and duplex DNA model systems.

    Science.gov (United States)

    Musumeci, Domenica; Rozza, Lucia; Merlino, Antonello; Paduano, Luigi; Marzo, Tiziano; Massai, Lara; Messori, Luigi; Montesarchio, Daniela

    2015-08-21

    The interaction of the anticancer Ru(iii) complex AziRu - in comparison with its analogue NAMI-A, currently in advanced clinical trials as an antimetastatic agent - with DNA model systems, both single stranded and duplex oligonucleotides, was investigated using a combined approach, including absorption UV-vis spectroscopy, circular dichroism (CD) and electrospray mass spectrometry (ESI-MS) techniques. UV-vis absorption spectra of the Ru complexes were recorded at different times in a pseudo-physiological solution, to monitor the ligand exchange processes in the absence and in the presence of the examined oligonucleotides. CD experiments provided information on the overall conformational changes of the DNA model systems induced by these metal complexes. UV- and CD-monitored thermal denaturation studies were performed to analyse the effects of AziRu and NAMI-A on the stability of the duplex structures. ESI-MS experiments, carried out on the oligonucleotide/metal complex mixtures under investigation, allowed us to detect the formation of stable adducts between the guanine-containing oligomers and the ruthenium complexes. These data unambiguously demonstrate that both AziRu and NAMI-A can interact with the DNA model systems. Although very similar in their structures, the two metal compounds manifest a markedly different reactivity with the examined sequences, respectively, with either a naked Ru(3+) ion or a Ru(Im)(3+) (Im = imidazole) fragment being incorporated into the oligonucleotide structure via stable linkages.

  12. Cytosolic RNA:DNA Duplexes Generated by Endogenous Reverse Transcriptase Activity as Autonomous Inducers of Skin Inflammation in Psoriasis.

    Science.gov (United States)

    Molès, Jean-Pierre; Griez, Anthony; Guilhou, Jean-Jacques; Girard, Céline; Nagot, Nicolas; Van de Perre, Philippe; Dujols, Pierre

    2017-01-01

    Psoriasis is a chronic skin disease of unknown ætiology. Recent studies suggested that a large amount of cytosolic DNA (cyDNA) in keratinocytes is breaking keratinocytes DNA tolerance and promotes self-sustained inflammation in the psoriatic lesion. We investigated the origin of this cyDNA. We show that, amongst all the possible DNA structures, the cyDNA could be present as RNA:DNA duplexes in keratinocytes. We further show that endogenous reverse transcriptase activities generate such duplexes and consequently activate the production of Th1-inflammatory cytokines. These observations open a new research avenue related to endogenous retroelements for the aetiology of psoriasis and probably of other human chronic inflammatory diseases.

  13. DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes

    Science.gov (United States)

    Smith, G. K.; Jie, J.; Fox, G. E.; Gao, X.

    1995-01-01

    DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.

  14. Parallel-stranded DNA and RNA duplexes - structural features and potential applications.

    Science.gov (United States)

    Szabat, Marta; Kierzek, Ryszard

    2017-12-01

    Nowadays, decades after the discovery of the right-handed B form of DNA, it is well known that nucleic acids have great conformational flexibility, exhibiting a large degree of variation in their structure. In nature, DNA and RNA exist in an antiparallel orientation, stabilized by Watson-Crick base pairs. However, in some cases, nucleic acid fragments with specific nucleotide sequences can adopt a parallel orientation involving non-canonical base pairing. Interestingly, parallel-stranded duplexes have been found in specific chromosome regions. Furthermore, parallel oriented regions have also been found in bacterial (Escherichia coli, Listeria innocua) and insect genomes (Drosophila melanogaster). These unusual structures could have a remarkable evolutionary role, as well as significant impact on biological processes. For example, parallel stretches were shown to be involved in processing the 3' ends of mRNAs and in specific gene silencing. Moreover, certain types of parallel-stranded duplexes may be useful tools, with several practical applications. They can constitute excellent templates for the formation of other structures and for the development of antigene and antisense approaches. © 2017 Federation of European Biochemical Societies.

  15. Optimized quantification of fragmented, free circulating DNA in human blood plasma using a calibrated duplex real-time PCR.

    Directory of Open Access Journals (Sweden)

    Martin Horlitz

    Full Text Available BACKGROUND: Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents. METHODOLOGY/PRINCIPAL FINDINGS: A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample. CONCLUSIONS: PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.

  16. Linker dependent intercalation of bisbenzimidazole-aminosugars in an RNA duplex; selectivity in RNA vs. DNA binding.

    Science.gov (United States)

    Ranjan, Nihar; Arya, Dev P

    2016-12-15

    Neomycin and Hoechst 33258 are two well-known nucleic acid binders that interact with RNA and DNA duplexes with high affinities respectively. In this manuscript, we report that covalent attachment of bisbenzimidazole unit derived from Hoechst 33258 to neomycin leads to intercalative binding of the bisbenzimidazole unit (oriented at 64-74° with respected to the RNA helical axis) in a linker length dependent manner. The dual binding and intercalation of conjugates were supported by thermal denaturation, CD, LD and UV-Vis absorption experiments. These studies highlight the importance of linker length in dual recognition by conjugates, for effective RNA recognition, which can lead to novel ways of recognizing RNA structures. Additionally, the ligand library screens also identify DNA and RNA selective compounds, with compound 9, containing a long linker, showing a 20.3°C change in RNA duplex T m with only a 13.0°C change in T m for the corresponding DNA duplex. Significantly, the shorter linker in compound 3 shows almost the reverse trend, a 23.8°C change in DNA T m , with only a 9.1°C change in T m for the corresponding RNA duplex. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex.

    Science.gov (United States)

    Zargarian, Loussiné; Ben Imeddourene, Akli; Gavvala, Krishna; Barthes, Nicolas P F; Michel, Benoit Y; Kenfack, Cyril A; Morellet, Nelly; René, Brigitte; Fossé, Philippe; Burger, Alain; Mély, Yves; Mauffret, Olivier

    2017-12-21

    Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out. Hence, a systematic structural and dynamic study is required to unveil the 3HCnt's limitations and to properly interpret the data obtained with this universal probe. Here, we investigated by NMR a 12-mer duplex, in which a central adenine was replaced by 3HCnt. The chemical shifts variations and nOe contacts revealed that the 3HCnt is well inserted in the DNA double helix with extensive stacking interactions with the neighbor base pairs. These observations are in excellent agreement with the steady-state and time-resolved fluorescence properties indicating that the 3HCnt fluorophore is protected from the solvent and does not exhibit rotational motion. The 3HCnt insertion in DNA is accompanied by the extrusion of the opposite nucleobase from the double helix. Molecular dynamics simulations using NMR-restraints demonstrated that 3HCnt fluorophore exhibits only translational dynamics. Taken together, our data showed an excellent intercalation of 3HCnt in the DNA double helix, which is accompanied by localized perturbations. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing.

  18. Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

    NARCIS (Netherlands)

    Etson, Candice M.; Hamdan, Samir M.; Richardson, Charles C.; Oijen, Antoine M. van; Richardson, Charles C.

    2010-01-01

    The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with

  19. Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

    Science.gov (United States)

    Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

    2017-04-01

    Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

  20. Recognition of base J in duplex DNA by J-binding protein

    NARCIS (Netherlands)

    Sabatini, Robert; Meeuwenoord, Nico; van Boom, Jacques H.; Borst, Piet

    2002-01-01

    beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding

  1. An exploration of sequence specific DNA-duplex/pyrene interactions for intercalated and surface-associated pyrene species. Final report, May 1, 1993--December 31, 1996

    Energy Technology Data Exchange (ETDEWEB)

    Netzel, T.L.

    1997-03-01

    The broad objective of this DOE sponsored work on photoinduced electron transfer (ET) within covalently modified DNA was to learn about the rates of Et among various DNA bases and commonly used organic electron donor (D) and acceptor (A) molecules. This hypothesis driven, multidisciplinary project combined skills in modified nucleic acid synthesis and in continuous and time-resolved optical spectroscopies. Covalently modified DNA chemistry as investigated in this program had two specific long term goals. The first was to use experimental and theoretical insights into the mechanisms of electron transfer (ET) reactions to design supramolecular assemblies of redox-active chromophores that function as efficient vectorial ET engines. The second was to construct oligonucleotide probes for real-time monitoring of intracellular processes involving DNA and RNA such as m-RNA expression and translocation. This research project laid the groundwork for studying ET reactions within DNA duplexes by examining the photophysics of uridine nucleosides which are covalently labeled at the 5-position with 1-pyrenyl chromophores.

  2. Restrained molecular dynamics of solvated duplex DNA using the particle mesh Ewald method

    International Nuclear Information System (INIS)

    Konerding, David E.; Cheatham, Thomas E.; Kollman, Peter A.; James, Thomas L.

    1999-01-01

    Restrained and unrestrained aqueous solution molecular dynamics simulations applying the particle mesh Ewald (PME) method to DNA duplex structures previously determined via in vacuo restrained molecular dynamics with NMR-derived restraints are reported. Without experimental restraints, the DNA decamer, d(CATTTGCATC).d(GATGCAAATG) and trisdecamer, d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), structures are stable on the nanosecond time scale and adopt conformations in the B-DNA family. These free DNA simulations exhibit behavior characteristic of PME simulations previously performed on DNA sequences, including a low helical twist, frequent sugar pucker transitions, BI- BII(ε-ζ) transitions and coupled crankshaft (α-γ) motion. Refinement protocols similar to the original in vacuo restrained molecular dynamics (RMD) refinements but in aqueous solution using the Cornell et al. force field [Cornell et al. (1995) J. Am. Chem. Soc., 117, 5179-5197] and a particle mesh Ewald treatment produce structures which fit the restraints very well and are very similar to the original in vacuo NMR structure, except for a significant difference in the average helical twist. Figures of merit for the average structure found in the RMD PME decamer simulations in solution are equivalent to the original in vacuo NMR structure while the figures of merit for the free MD simulations are significantly higher. The free MD simulations with the PME method, however, lead to some sequence-dependent structural features in common with the NMR structures, unlike free MD calculations with earlier force fields and protocols. There is some suggestion that the improved handling of electrostatics by PME improves long-range structural aspects which are not well defined by the short-range nature of NMR restraints

  3. High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions

    International Nuclear Information System (INIS)

    Chou, Shanho; Flynn, P.; Wang, A.; Reid, B.

    1991-01-01

    Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(AACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but these results differ somewhat from previous theoretical model studies

  4. DNA Duplex Length and Salt Concentration Dependence of Enthalpy−Entropy Compensation Parameters for DNA Melting

    KAUST Repository

    Starikov, E. B.

    2009-08-20

    Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field. © 2009 American Chemical Society.

  5. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    Science.gov (United States)

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  6. Crystal structure of RNA-DNA duplex provides insight into conformational changes induced by RNase H binding.

    Science.gov (United States)

    Davis, Ryan R; Shaban, Nadine M; Perrino, Fred W; Hollis, Thomas

    2015-01-01

    RNA-DNA hybrids play essential roles in a variety of biological processes, including DNA replication, transcription, and viral integration. Ribonucleotides incorporated within DNA are hydrolyzed by RNase H enzymes in a removal process that is necessary for maintaining genomic stability. In order to understand the structural determinants involved in recognition of a hybrid substrate by RNase H we have determined the crystal structure of a dodecameric non-polypurine/polypyrimidine tract RNA-DNA duplex. A comparison to the same sequence bound to RNase H, reveals structural changes to the duplex that include widening of the major groove to 12.5 Å from 4.2 Å and decreasing the degree of bending along the axis which may play a crucial role in the ribonucleotide recognition and cleavage mechanism within RNase H. This structure allows a direct comparison to be made about the conformational changes induced in RNA-DNA hybrids upon binding to RNase H and may provide insight into how dysfunction in the endonuclease causes disease.

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

    Science.gov (United States)

    Lomzov, Alexander A; Vorobjev, Yury N; Pyshnyi, Dmitrii V

    2015-12-10

    A molecular dynamics simulation approach was applied for the prediction of the thermal stability of oligonucleotide duplexes. It was shown that the enthalpy of the DNA/DNA complex formation could be calculated using this approach. We have studied the influence of various simulation parameters on the secondary structure and the hybridization enthalpy value of Dickerson-Drew dodecamer. The optimal simulation parameters for the most reliable prediction of the enthalpy values were determined. The thermodynamic parameters (enthalpy and entropy changes) of a duplex formation were obtained experimentally for 305 oligonucleotides of various lengths and GC-content. The resulting database was studied with molecular dynamics (MD) simulation using the optimized simulation parameters. Gibbs free energy changes and the melting temperatures were evaluated using the experimental correlation between enthalpy and entropy changes of the duplex formation and the enthalpy values calculated by the MD simulation. The average errors in the predictions of enthalpy, the Gibbs free energy change, and the melting temperature of oligonucleotide complexes were 11%, 10%, and 4.4 °C, respectively. We have shown that the molecular dynamics simulation gives a possibility to calculate the thermal stability of native DNA/DNA complexes a priori with an unexpectedly high accuracy.

  8. Cisplatin adducts on a GGG sequence within a DNA duplex studied by NMR spectroscopy and molecular dynamics simulations.

    Science.gov (United States)

    Téletchéa, Stéphane; Skauge, Tormod; Sletten, Einar; Kozelka, Jirí

    2009-11-16

    The antitumor drug cisplatin(cis-[PtCl2(NH3)2]) reacts with cellular DNA to form GG intrastrand adducts between adjacent guanines as predominant lesions. GGG sites have been shown to be hotspots of platination. To study the structural perturbation induced by binding of cisplatin to two adjacent guanines of a GGG trinucleotide,we examined here the decanucleotide duplex d[(G1C2C3G*4 G*5 G6T7-C8G9C10).d(G11C12G13A14C15C16C17G18-G19C20)] (dsCG*G*G) intrastrand cross-linked at the G* guanines by cis-{Pt(NH3)2}2+ using NMR spectroscopy and molecular dynamics (MD) simulations.The NMR spectra of dsCG*G*G were found to be similar to those of previously characterized DNA duplexes cross-linked by cisplatin at apyG*G*X site (py=pyrimidine; X=C,T, A). This similarity of NMR spectra indicates that the base at the 3'-side of the G*G*-Pt cross-link does not affect the structure to a large extent. An unprecedented reversible isomerization between the duplex dsCG*G*G (bearing a G*4 G*5 -Pt chelate) and duplex dsGG*G*T (bearing a G*5 G*6 -Pt chelate)was observed, which yielded a 40:60 equilibrium between the two intrastrand GG-Pt cross-links. No formation of interstrand cross-links was observed.NMR spectroscopic data of dsCG*G*G indicated that the deoxyribose of the 5'-G* adopts an N-type conformation, and the cytidines C3, C15,and C16 have average phase angles intermediate between S and N. The NMR spectroscopic chemical shifts of dsGG*G*T showed some fundamental differences to those of pyG*G*-platinum adducts but were in agreement with the NMR spectra reported previously for the DNA duplexes crosslinked at an AG*G*C sequence by cisplatin or oxaliplatin. The presence of apurine instead of a pyrimidine at the 5'-side of the G*G* cross-link seems therefore to affect the structure of the XG* step significantly.

  9. Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA.

    Science.gov (United States)

    Yang, Changwon; Kim, Eunae; Pak, Youngshang

    2015-09-18

    Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson-Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine-thymine (A-T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

    Directory of Open Access Journals (Sweden)

    CHENG Fang

    2013-04-01

    Full Text Available Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

  11. Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes

    Science.gov (United States)

    2011-01-01

    Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand–duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form. PMID:21928795

  12. Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry. Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy

    DEFF Research Database (Denmark)

    Sigurdsson, Snorri; Vogel, Stefan; Shelke, Sandip

    2010-01-01

    This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis...

  13. DNA duplex stability as discriminative characteristic for Escherichia coli σ(54)- and σ(28)- dependent promoter sequences.

    Science.gov (United States)

    de Avila e Silva, Scheila; Forte, Franciele; T S Sartor, Ivaine; Andrighetti, Tahila; J L Gerhardt, Günther; Longaray Delamare, Ana Paula; Echeverrigaray, Sergio

    2014-01-01

    The advent of modern high-throughput sequencing has made it possible to generate vast quantities of genomic sequence data. However, the processing of this volume of information, including prediction of gene-coding and regulatory sequences remains an important bottleneck in bioinformatics research. In this work, we integrated DNA duplex stability into the repertoire of a Neural Network (NN) capable of predicting promoter regions with augmented accuracy, specificity and sensitivity. We took our method beyond a simplistic analysis based on a single sigma subunit of RNA polymerase, incorporating the six main sigma-subunits of Escherichia coli. This methodology employed successfully re-discovered known promoter sequences recognized by E. coli RNA polymerase subunits σ(24), σ(28), σ(32), σ(38), σ(54) and σ(70), with highlighted accuracies for σ(28)- and σ(54)- dependent promoter sequences (values obtained were 80% and 78.8%, respectively). Furthermore, the discrimination of promoters according to the σ factor made it possible to extract functional commonalities for the genes expressed by each type of promoter. The DNA duplex stability rises as a distinctive feature which improves the recognition and classification of σ(28)- and σ(54)- dependent promoter sequences. The findings presented in this report underscore the usefulness of including DNA biophysical parameters into NN learning algorithms to increase accuracy, specificity and sensitivity in promoter beyond what is accomplished based on sequence alone. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  14. Structural effects of internucleotide linkage modification at mismatch site in DNA/RNA duplex: Raman study

    Czech Academy of Sciences Publication Activity Database

    Kříž, M.; Němeček, D.; Turpin, P. Y.; Rosenberg, Ivan; Štěpánek, J.

    2008-01-01

    Roč. 48, č. 1 (2008), s. 148-152 ISSN 0924-2031 R&D Projects: GA ČR GA203/05/0827 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleic acid * mismatch * hybrid duplex * Raman spectroscopy Subject RIV: CC - Organic Chemistry Impact factor: 1.810, year: 2008

  15. In Vivo Screening of Chemically Modified RNA duplexes for their Ability to Induce Innate Immune Responses

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    Due to their sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...... protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form...

  16. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.

    Science.gov (United States)

    Félix-Urquídez, Dalmira; Pérez-Urquiza, Melina; Valdez Torres, José-Benigno; León-Félix, Josefina; García-Estrada, Raymundo; Acatzi-Silva, Abraham

    2016-01-05

    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio.

  17. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize

    Science.gov (United States)

    2015-01-01

    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%–100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio. PMID:26605751

  18. Positive cooperativity of the specific binding between Hg2+ ion and T:T mismatched base pairs in duplex DNA

    International Nuclear Information System (INIS)

    Torigoe, Hidetaka; Miyakawa, Yukako; Ono, Akira; Kozasa, Tetsuo

    2012-01-01

    Highlights: ► Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio. ► The binding constant between Hg 2+ and the T:T mismatched base pair was 10 6 M −1 . ► The binding constant was larger than those for nonspecific metal–DNA interactions. ► The binding constant for the second Hg 2+ was larger than that for the first Hg 2+ . ► The positive cooperative binding was observed between Hg 2+ and multiple T:T. - Abstract: Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides have been developed for their potential applications in nanotechnology. We recently found that a natural T:T mismatched base pair bound with Hg 2+ ion to form a novel T–Hg–T base pair. Here, we examined the thermodynamic properties of the binding between Hg 2+ and each of the single and double T:T mismatched base pair duplex DNAs by isothermal titration calorimetry. Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio with 10 6 M −1 binding constant, which was significantly larger than those for nonspecific metal ion–DNA interactions. In the Hg 2+ –double T:T mismatched base pair interaction, the affinity for the second Hg 2+ binding was significantly larger than that for the first Hg 2+ binding. The positively cooperative binding may be favorable to align multiple Hg 2+ in duplex DNA for the application of the metal-mediated base pairs in nanotechnology.

  19. 2'-O-methyl-modified anti-MDR1 fork-siRNA duplexes exhibiting high nuclease resistance and prolonged silencing activity.

    Science.gov (United States)

    Petrova Kruglova, Natalya S; Meschaninova, Mariya I; Venyaminova, Alya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2010-12-01

    The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.

  20. An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

    Science.gov (United States)

    Eisenberg, S; Scott, J F; Kornberg, A

    1976-05-01

    Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.

  1. Magnetic studies of Co2+, Ni2+, and Zn2+-modified DNA double-crossover lattices

    Science.gov (United States)

    Dugasani, Sreekantha Reddy; Oh, Young Hoon; Gnapareddy, Bramaramba; Park, Tuson; Kang, Won Nam; Park, Sung Ha

    2018-01-01

    We fabricated divalent-metal-ion-modified DNA double-crossover (DX) lattices on a glass substrate and studied their magnetic characteristics as a function of ion concentrations [Co2+], [Ni2+] and [Zn2+]. Up to certain critical concentrations, the DNA DX lattices with ions revealed discrete S-shaped hysteresis, i.e. characteristics of strong ferromagnetism, with significant changes in the coercive field, remanent magnetization, and susceptibility. Induced magnetic dipoles formed by metal ions in DNA duplex in the presence of a magnetic field imparted ferromagnetic behaviour. By considering hysteresis and the magnitude of magnetization in a magnetization-magnetic field curve, Co2+-modified DNA DX lattices showed a relatively strong ferromagnetic nature with an increasing (decreasing) trend of coercive field and remanent magnetization when [Co2+] ≤ 1 mM ([Co2+] > 1 mM). In contrast, Ni2+ and Zn2+-modified DNA DX lattices exhibited strong and weak ferromagnetic behaviours at lower (≤1 mM for Ni2+ and ≤0.5 mM for Zn2+) and higher (>1 mM for Ni2+ and >0.5 mM for Zn2+) concentrations of ions, respectively. About 1 mM of [Co2+], [Ni2+] and [Zn2+] in DNA DX lattices was of special interest with regard to physical characteristics and was identified to be an optimum concentration of each ion. Finally, we measured the temperature-dependent magnetic characteristics of the metal-ion-modified DNA DX lattices. Nonzero magnetization and inverse susceptibility with almost constant values were observed between 25 and 300 K, with no indication of a magnetic transition. This indicated that the magnetic Curie temperatures of Co2+, Ni2+ and Zn2+-modified DNA DX lattices were above 300 K.

  2. Oligonucleotides containing a piperazino-modified 2'-amino-LNA monomer exhibit very high duplex stability and remarkable nuclease resistance

    DEFF Research Database (Denmark)

    Lou, Chenguang; Vester, Birte; Wengel, Jesper

    2015-01-01

    Incorporation of a piperazino-modified 2'-amino-LNA monomer (PipLNA-T) into oligonucleotides conferred very high affinity and base-pairing selectivity towards complementary DNA and RNA strands. Furthermore, one PipLNA-T modification provided a robust nuclease resistance that safeguarded three...

  3. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    Science.gov (United States)

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  4. Structure of the 1,N2-etheno-2'-deoxyguanosine adduct in duplex DNA at pH 8.6.

    Science.gov (United States)

    Shanmugam, Ganesh; Goodenough, Angela K; Kozekov, Ivan D; Guengerich, F Peter; Rizzo, Carmelo J; Stone, Michael P

    2007-11-01

    The structure of the 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondG) adduct, arising from the reaction of vinyl chloride with dG, was determined in the oligonucleotide duplex 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCCATGCG)-3' (X=1,N(2)-epsilondG) at pH 8.6 using high resolution NMR spectroscopy. The exocyclic lesion prevented Watson-Crick base-pairing capability at the adduct site and resulted in an approximately 17 degrees C decrease in Tm of the oligodeoxynucleotide duplex. At neutral pH, conformational exchange resulted in spectral line broadening near the adducted site, and it was not possible to determine the structure. However, at pH 8.6, it was possible to obtain well-resolved (1)H NMR spectra. This enabled a total of 385 NOE-based distance restraints to be obtained, consisting of 245 intra- and 140 inter-nucleotide distances. The (31)P NMR spectra exhibited two downfield-shifted resonances, suggesting a localized perturbation of the DNA backbone. The two downfield (31)P resonances were assigned to G(7) and C(19). The solution structure was refined by molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints, using a simulated annealing protocol. The generalized Born approximation was used to simulate solvent. The emergent structures indicated that the 1,N(2)-epsilondG-induced structural perturbation was localized at the X(6).C(19) base pair, and its 5'-neighbor T(5).A(20). Both 1,N(2)-epsilondG and the complementary dC adopted the anti conformation about the glycosyl bonds. The 1,N (2)-epsilondG adduct was inserted into the duplex but was shifted towards the minor groove as compared to dG in a normal Watson-Crick C.G base pair. The complementary cytosine was displaced toward the major groove. The 5'-neighbor T(5).A(20) base pair was destabilized with respect to Watson-Crick base pairing. The refined structure predicted a bend in the helical axis associated with the adduct site.

  5. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Directory of Open Access Journals (Sweden)

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  6. G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

    Science.gov (United States)

    Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

    2016-10-01

    The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

  7. Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

    Directory of Open Access Journals (Sweden)

    Ineke Brouwer

    2017-03-01

    Full Text Available Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA. In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA, interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

  8. Solution structure of the DNA decamer duplex containing a 3′-T·T base pair of the cis–syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis–syn dimer

    OpenAIRE

    Lee, Joon-Hwa; Choi, Yun-Jeong; Choi, Byong-Seok

    2000-01-01

    The cis–syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis–syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3′-T of the cis–syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3′-T·T base pair of the cis–syn dimer had base pair geometry that w...

  9. Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes.

    Science.gov (United States)

    Anosova, Irina; Kowal, Ewa A; Sisco, Nicholas J; Sau, Sujay; Liao, Jen-Yu; Bala, Saikat; Rozners, Eriks; Egli, Martin; Chaput, John C; Van Horn, Wade D

    2016-09-15

    Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four-carbon threose backbone in place of the five-carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson-Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base-pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A-like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric "breathing" fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Pyrene-modified unlocked nucleic acids: synthesis, thermodynamic studies, and fluorescent properties

    DEFF Research Database (Denmark)

    Karlsen, Kasper K; Pasternak, Anna; Jensen, Troels B

    2012-01-01

    Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene...... pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools....

  11. Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

    DEFF Research Database (Denmark)

    Izvolsky, K I; Demidov, V V; Nielsen, P E

    2000-01-01

    of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable......I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...

  12. Thermodynamic stability and energetics of DNA duplexes containing major intrastrand cross-links of second-generation antitumor dinuclear Pt(II) complexes.

    Science.gov (United States)

    Florian, Jakub; Kasparkova, Jana; Farrell, Nicholas P; Brabec, Viktor

    2012-02-01

    The effects of major DNA intrastrand cross-links of antitumor dinuclear Pt(II) complexes [{trans-PtCl(NH(3))(2)}(2)-μ-{trans-(H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (1) and [{PtCl(DACH)}(2)-μ-{H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (2) (DACH is 1,2-diaminocyclohexane) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes containing the single 1,2, 1,3, or 1,5 intrastrand cross-links at guanine residues in the central TGGT, TGTGT, or TGTTTGT sequences, respectively, were prepared and analyzed by differential scanning calorimetry. The unfolding of the platinated duplexes was accompanied by unfavorable free energy terms. The efficiency of the cross-links to thermodynamically destabilize the duplex depended on the number of base pairs separating the platinated bases. The trend was 1,5→1,2→1,3 cross-link of 1 and 1,5→1,3→1,2 cross-link of 2. Interestingly, the results showed that the capability of the cross-links to reduce the thermodynamic stability of DNA (ΔG(298)(0)) correlated with the extent of conformational distortions induced in DNA by various types of intrastrand cross-links of 1 or 2 determined by chemical probes of DNA conformation. We also examined the efficiency of the mammalian nucleotide excision repair systems to remove from DNA the intrastrand cross-links of 1 or 2. The efficiency of the excinucleases to remove the cross-links from DNA depended on the length of the cross-link; the trend was identical to that observed for the efficiency of the intrastrand cross-links to thermodynamically destabilize the duplex. Thus, the results are consistent with the thesis that an important factor that determines the susceptibility of the intrastrand cross-links of dinuclear platinum complexes 1 and 2 to be removed from DNA by nucleotide excision repair is the efficiency of these lesions to thermodynamically destabilize DNA.

  13. NMR spectroscopic studies on metallo-base-pair in DNA duplex

    Czech Academy of Sciences Publication Activity Database

    Tanaka, Y.; Okamoto, I.; Furuita, K.; Šebera, Jakub; Kondo, J.; Torigoe, H.; Urata, H.; Dairaku, T.; Ono, A.; Kojima, C.; Sychrovský, Vladimír

    2014-01-01

    Roč. 19, Suppl 2 (2014), S732-S733 ISSN 0949-8257. [European Biological Inorganic Chemistry Conference /12./. 24.08.2014-28.08.2014, Zurich] Institutional support: RVO:61388963 Keywords : NMR * DNA * mercury * thymine Subject RIV: CF - Physical ; Theoretical Chemistry

  14. Thermal stability and energetics of 15-mer DNA duplex interstrand crosslinked by trans-diamminedichloroplatinum(II)

    Czech Academy of Sciences Publication Activity Database

    Hofr, Ctirad; Brabec, Viktor

    2005-01-01

    Roč. 77, č. 4 (2005), s. 222-229 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GP202/01/D110; GA ČR(CZ) GA305/02/1552; GA AV ČR(CZ) LZ1K03010 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA * interstrand cross-link * transplatin Subject RIV: BO - Biophysics Impact factor: 2.545, year: 2005

  15. Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis.

    Science.gov (United States)

    Angevine, Christopher E; Seashols-Williams, Sarah J; Reiner, Joseph E

    2016-03-01

    Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture.

  16. Chemically modified DNA : methods and applications

    NARCIS (Netherlands)

    Gjonaj, Lorina

    2013-01-01

    Over het toepassen van DNA in de chemie DNA is een fascinerend biopolymeer dat gemakkelijk gemanipuleerd kan worden. Doel van het onderzoek van Lorina Gjonaj was het ontwikkelen van nieuwe methodologie om DNA chemisch te modificeren. Verschillende functionaliteiten zijn aan DNA gekoppeld. Deze

  17. Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components

    Directory of Open Access Journals (Sweden)

    Marian Filipiak

    2008-03-01

    Full Text Available An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5’-phosphate end to amino group of cysteamine self-assembled monolayer (SAM on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3’- dimethylaminopropyl-carbodiimide (EDC and N-hydroxy-sulfosuccinimide (NHS. The hybridization reaction on the electrode surface was detected via methylene blue (MB presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs give a broad perspectives for analytical application of the biosensor.

  18. Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study

    Science.gov (United States)

    Pan, Feng; Roland, Christopher; Sagui, Celeste

    2014-01-01

    The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5′-CGCGCGCGCGCG-3′)2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na+, K+ and Mg2 +, with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6]2+ ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6]2+ to distant phosphates, resulting in acute bending of A-RNA; tight ‘ion traps’ in Z-RNA between C-O2 and the C-O2′ atoms in GpC steps; and others. PMID:25428372

  19. Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

    OpenAIRE

    Pande, Vineet; Nilsson, Lennart

    2008-01-01

    Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–D...

  20. Water-bridged hydrogen bond formation between 5-hydroxylmethylcytosine (5-hmC) and its 3'-neighbouring bases in A- and B-form DNA duplexes.

    Science.gov (United States)

    Wang, Rui; Ranganathan, Srivathsan V; Valsangkar, Vibhav A; Magliocco, Stephanie M; Shen, Fusheng; Chen, Alan; Sheng, Jia

    2015-11-25

    5-Hydroxylmethylcytosine (5hmC) has been recognized as the sixth base with important biological functions in many tissues and cell types. We present here the high-resolution crystal structures and molecular simulation studies of both A-form and B-form DNA duplexes containing 5hmC. We observed that 5hmC interacts with its 3'-neighboring bases through water-bridged hydrogen bonds and these interactions may affect the further oxidation of 5hmC.

  1. Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

    Science.gov (United States)

    Ahour, F; Shamsi, A

    2017-09-01

    Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

    Science.gov (United States)

    Itou, Hiroshi; Yagura, Masaru; Shirakihara, Yasuo; Itoh, Tateo

    2015-02-06

    Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Topological solitons in DNA with modified potential

    Directory of Open Access Journals (Sweden)

    E Behjat

    2010-06-01

    Full Text Available DNA is not only an essential research subject for biologists, but also it raises very interesting questions for physicists.The open states in DNA double helix can lead to topological solitons. Since DNA is a very long molecule of order a meter or so long and nano-scale width, solitons can propagate along the molecule. In this paper, considering a correction term in the interaction potential between two chains, we study the dispersion relation analytically, and obtain the soliton solutions using a new relaxation method. Then we compare our solutions and its energy with those obtained by others without the proposed correction term.

  4. Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.

    Science.gov (United States)

    Rosenblum, Sydney L; Weiden, Aurora G; Lewis, Eliza L; Ogonowsky, Alexie L; Chia, Hannah E; Barrett, Susanna E; Liu, Mira D; Leconte, Aaron M

    2017-04-18

    Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Complex structure of the DNA-binding domain of AdpA, the global transcription factor in Streptomyces griseus, and a target duplex DNA reveals the structural basis of its tolerant DNA sequence specificity.

    Science.gov (United States)

    Yao, Ming Dong; Ohtsuka, Jun; Nagata, Koji; Miyazono, Ken-Ichi; Zhi, Yuehua; Ohnishi, Yasuo; Tanokura, Masaru

    2013-10-25

    AdpA serves as the global transcription factor in the A-factor regulatory cascade, controlling the secondary metabolism and morphological differentiation of the filamentous bacterium Streptomyces griseus. AdpA binds to over 500 operator regions with the consensus sequence 5'-TGGCSNGWWY-3' (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To elucidate the structural basis of this tolerant DNA recognition by AdpA, we focused on the interaction between the DNA-binding domain of AdpA (AdpA-DBD), which consists of two helix-turn-helix motifs, and a target duplex DNA containing the consensus sequence 5'-TGGCGGGTTC-3'. The crystal structure of the AdpA-DBD-DNA complex and the mutant analysis of AdpA-DBD revealed its unique manner of DNA recognition, whereby only two arginine residues directly recognize the consensus sequence, explaining the strict recognition of G and C at positions 2 and 4, respectively, and the tolerant recognition of other positions of the consensus sequence. AdpA-DBD confers tolerant DNA sequence specificity to AdpA, allowing it to control hundreds of genes as a global transcription factor.

  6. Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

    Directory of Open Access Journals (Sweden)

    Harald Niederstätter

    Full Text Available BACKGROUND: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA, sequence variation in particular mt haplogroups (hgs and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples or the derived 7028C allele (hg H samples in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA. The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the

  7. Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.

    Science.gov (United States)

    Chen, Dan; Pan, Shiyang; Xie, Erfu; Gao, Li; Xu, Huaguo; Xia, Wenying; Xu, Ting; Huang, Peijun

    2017-01-01

    Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, PDNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, PDNA, showing promising application in clinical diagnosis.

  8. Duplex structural differences and not 2′-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA

    OpenAIRE

    Olimpo, Jeffrey T.; DeStefano, Jeffrey J.

    2010-01-01

    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA–DNA versus DNA–DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3′ recessed DNA base) are required for stable binding to RNA–DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA–DNA. Replacing 2′- hydroxyls on pivotal RNA bases with 2′-O-methyls did not affect stability, indicating that interactions between hy...

  9. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    Science.gov (United States)

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  10. Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products.

    Science.gov (United States)

    López-Calleja, Inés María; de la Cruz, Silvia; Martín, Rosario; González, Isabel; García, Teresa

    2015-01-01

    The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg(-1) for sesame and 1.4 mg kg(-1) for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.

  11. The effects of a neutral cosolute on the B to Z transition for DNA duplexes incorporating both CG and CA steps.

    Science.gov (United States)

    Phromsiri, Pakinee; Gerling, Rebecca R; Blose, Joshua M

    2017-11-02

    In the cell, nearly 40% of the volume is occupied by macromolecular crowding agents and smaller osmolytes accumulate in response to environmental stresses. Of particular interest is the influence of osmolytes on the transition of the right-handed B-DNA to the left-handed Z-DNA. Due to the correlation between Z-DNA formation potential and regions of active transcription, Z-DNA is believed to serve a vital role in the transcription process, and changes in osmolyte concentration may influence transcription as a part of the stress response. We utilized circular dichroism spectroscopy to monitor changes in conformation of DNA duplexes containing a full-turn of Z-DNA in the presence and absence of PEG 200. We used PEG 200 as a model neutral cosolute. Sodium ion titrations revealed that PEG 200 influenced the folding of Z-DNA compared to dilute solution conditions by decreasing the free energy of folding, increasing folding cooperativity, and decreasing the in vitro [Na + ] and Δn required for folding for all sequences tested, even those that included 40% CA steps instead of the classic CG repeats. Moreover, the presence of 40% PEG 200 induced the Z-form conformation in sequences that would not fully adopt the Z-form structure even in 5 M NaCl. These results suggest that osmolytes may play a significant role in supporting the transient formation of Z-DNA in vivo, and that sequences containing a significant amounts of CA instead of CG repeats may more favorably adopt the Z-conformation as a part of binding and regulatory processes than had been previously considered.

  12. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

  13. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    Science.gov (United States)

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

  14. Quantification of Chemical and Mechanical Effects on the Formation of the G-Quadruplex and i-Motif in Duplex DNA.

    Science.gov (United States)

    Selvam, Sangeetha; Mandal, Shankar; Mao, Hanbin

    2017-09-05

    The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription.

  15. NMR structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) comprising the tRNA-DNA junction formed during initiation of HIV-1 reverse transcription

    International Nuclear Information System (INIS)

    Szyperski, Thomas; Goette, Matthias; Billeter, Martin; Perola, Emanuele; Cellai, Luciano; Heumann, Hermann; Wuethrich, Kurt

    1999-01-01

    A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of ∼11 A and ∼8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex

  16. Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts

    NARCIS (Netherlands)

    Sabatini, Robert; Meeuwenoord, Nico; van Boom, Jacques H.; Borst, Piet

    2002-01-01

    Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a

  17. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

    Vester, Birte; Boel, Anne Marie; Lobedanz, Sune

    2008-01-01

    Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly...... in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage....

  18. Modified Classical Graph Algorithms for the DNA Fragment Assembly Problem

    Directory of Open Access Journals (Sweden)

    Guillermo M. Mallén-Fullerton

    2015-09-01

    Full Text Available DNA fragment assembly represents an important challenge to the development of efficient and practical algorithms due to the large number of elements to be assembled. In this study, we present some graph theoretical linear time algorithms to solve the problem. To achieve linear time complexity, a heap with constant time operations was developed, for the special case where the edge weights are integers and do not depend on the problem size. The experiments presented show that modified classical graph theoretical algorithms can solve the DNA fragment assembly problem efficiently.

  19. Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

    Science.gov (United States)

    Kathuria, Preetleen; Sharma, Purshotam; Manderville, Richard A; Wetmore, Stacey D

    2017-08-21

    Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C 8 -dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C 8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C 8 -dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

  20. Amino acids attached to 2'-amino-LNA: Synthesis of DNA mixmer oligonucleotides with increased duplex stability

    DEFF Research Database (Denmark)

    Johannsen, Marie Willaing; Wengel, Jesper; Wamberg, Michael Chr.

    2010-01-01

    -LNA nucleosides derivatized with amino acids have been synthesized and incorporated into DNA oligonucleotides. Following oligonucleotide synthesis, peptides have been added using solid phase peptide coupling chem. Modification of oligonucleotides with pos. charged residues greatly improves thermal stability....

  1. Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

    Science.gov (United States)

    Tripathi, Prashant; Moinuddin; Dixit, Kiran; Mir, Abdul Rouf; Habib, Safia; Alam, Khursheed; Ali, Asif

    2014-07-01

    Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

    International Nuclear Information System (INIS)

    Bhattarai, Shanta Raj; Remant Bahadur, K.C.; Aryal, Santosh; Bhattarai, Narayan; Kim, Sun Young; Yi, Ho Keun; Hwang, Pyoung Han; Kim, Hak Yong

    2008-01-01

    Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10∼12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0∼66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher β-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin (registered) ) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery

  3. Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

    Energy Technology Data Exchange (ETDEWEB)

    Bhattarai, Shanta Raj; Remant Bahadur, K.C.; Aryal, Santosh [Chonbuk National University, Department of Bionanosystem Engineering (Korea, Republic of); Bhattarai, Narayan [University of Washington, Department of Materials Science and Engineering (United States); Kim, Sun Young [Chonbuk National University, Department of Pediatrics, School of Medicine (Korea, Republic of); Yi, Ho Keun [Chonbuk National University, Department of Biochemistry, School of Dentisty (Korea, Republic of); Hwang, Pyoung Han [Chonbuk National University, Department of Pediatrics, School of Medicine (Korea, Republic of); Kim, Hak Yong [Chonbuk National University, Department of Textile Engineering (Korea, Republic of)], E-mail: khy@moak.chonbuk.ac.kr

    2008-01-15

    Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10{approx}12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0{approx}66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher {beta}-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin (registered) ) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery.

  4. Thermodynamic and structural properties of the specific binding between Ag⁺ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair.

    Science.gov (United States)

    Torigoe, Hidetaka; Okamoto, Itaru; Dairaku, Takenori; Tanaka, Yoshiyuki; Ono, Akira; Kozasa, Tetsuo

    2012-11-01

    Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences. Copyright © 2012 Elsevier

  5. Bis-pyrene-modified unlocked nucleic acids: synthesis, hybridization studies, and fluorescent properties

    DEFF Research Database (Denmark)

    Perlíková, Pavla; Ejlersen, Maria; Langkjaer, Niels

    2014-01-01

    Efficient synthesis of a building block for the incorporation of a bis-pyrene-modified unlocked nucleic acid (UNA) into oligonucleotides (DNA*) was developed. The presence of bis-pyrene-modified UNA within a duplex leads to duplex destabilization that is more profound in DNA*/RNA and less distinct......)uracil:pyrene exciplex emission in the single-stranded form. Such fluorescent properties enable the application of bis-pyrene-modified UNA in the development of fluorescence probes for DNA/RNA detection and for detection of deletions at specific positions....

  6. DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr [Department of Life Sciences, University of Ulsan, Ulsan (Korea, Republic of); Bohr, Vilhelm A. [Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, Baltimore, MD (United States)

    2011-08-12

    Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

  7. Random amplified polymorphic DNA analysis of genetically modified organisms.

    Science.gov (United States)

    Yoke-Kqueen, Cheah; Radu, Son

    2006-12-15

    Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.

  8. The Effect of Small Cosolutes that Mimic Molecular Crowding Conditions on the Stability of Triplexes Involving Duplex DNA

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2016-02-01

    Full Text Available Triplex stability is studied in crowding conditions using small cosolutes (ethanol, acetonitrile and dimethylsulfoxide by ultraviolet (UV, circular dichroism (CD and nuclear magnetic resonance (NMR spectroscopies. The results indicate that the triplex is formed preferentially when the triplex forming oligonucleotide (TFO is RNA. In addition, DNA triplexes (D:D·D are clearly less stable in cosolute solutions while the stability of the RNA triplexes (R:D·D is only slightly decreased. The kinetic of triplex formation with RNA-TFO is slower than with DNA-TFO and the thermal stability of the triplex is increased with the salt concentration in EtOH-water solutions. Accordingly, RNA could be considered a potential molecule to form a stable triplex for regulatory purposes in molecular crowding conditions.

  9. [Detection of recombinant DNA from genetically modified papaya].

    Science.gov (United States)

    Goda, Y; Asano, T; Shibuya, M; Hino, A; Toyoda, M

    2001-08-01

    A method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) papaya (55-1 line), of which the mandatory safety assessment has not been finished in Japan because of insufficient data. The papaya intrinsic papain gene was used as an internal control. The results of PCR amplification of the papain gene segment indicated that a commercial silica membrane type kit (QIAGEN DNeasy plant mini) was useful for extraction of DNA from papaya fruit, but not for extraction from canned papaya fruit. On the other hand, a commercial ion-exchange type kit (QIAGEN Genomic-tip) provided enough purified DNA for PCR from canned papaya fruit. Compared with the parental line and other commercial non-GM papayas, the DNA from GM papaya fruit provided specific amplification bands in PCR with five primer pairs (Nos. 2-6) including beta-glucuronidase and neomycin phosphotransferase II gene-specific ones. On the other hand, the primer pairs recognizing these genes showed false-positive results when we used DNAs from canned papaya. Therefore, we recommend that the primer pairs (Nos. 5 and 6) recognizing the sequences derived from two different species of organism should be used in order to detect specifically the GM papaya in canned fruits.

  10. Study of Photochemical Cytosine to Uracil Transition via Ultrafast Photo-Cross-Linking Using Vinylcarbazole Derivatives in Duplex DNA.

    Science.gov (United States)

    Sethi, Siddhant; Nakamura, Shigetaka; Fujimoto, Kenzo

    2018-04-04

    Gene therapies, including genome editing, RNAi, anti-sense technology and chemical DNA editing are becoming major methods for the treatment of genetic disorders. Techniques like CRISPR-Cas9, zinc finger nuclease (ZFN) and transcription activator-like effector-based nuclease (TALEN) are a few such enzymatic techniques. Most enzymatic genome editing techniques have their disadvantages. Thus, non-enzymatic and non-invasive technologies for nucleic acid editing has been reported in this study which might possess some advantages over the older methods of DNA manipulation. 3-cyanovinyl carbazole ( CNV K) based nucleic acid editing takes advantage of photo-cross-linking between a target pyrimidine and the CNV K to afford deamination of cytosine and convert it to uracil. This method previously required the use of high temperatures but, in this study, it has been optimized to take place at physiological conditions. Different counter bases (inosine, guanine and cytosine) complementary to the target cytosine were used, along with derivatives of CNV K ( NH2V K and OHV K) to afford the deamination at physiological conditions.

  11. Study of Photochemical Cytosine to Uracil Transition via Ultrafast Photo-Cross-Linking Using Vinylcarbazole Derivatives in Duplex DNA

    Directory of Open Access Journals (Sweden)

    Siddhant Sethi

    2018-04-01

    Full Text Available Gene therapies, including genome editing, RNAi, anti-sense technology and chemical DNA editing are becoming major methods for the treatment of genetic disorders. Techniques like CRISPR-Cas9, zinc finger nuclease (ZFN and transcription activator-like effector-based nuclease (TALEN are a few such enzymatic techniques. Most enzymatic genome editing techniques have their disadvantages. Thus, non-enzymatic and non-invasive technologies for nucleic acid editing has been reported in this study which might possess some advantages over the older methods of DNA manipulation. 3-cyanovinyl carbazole (CNVK based nucleic acid editing takes advantage of photo-cross-linking between a target pyrimidine and the CNVK to afford deamination of cytosine and convert it to uracil. This method previously required the use of high temperatures but, in this study, it has been optimized to take place at physiological conditions. Different counter bases (inosine, guanine and cytosine complementary to the target cytosine were used, along with derivatives of CNVK (NH2VK and OHVK to afford the deamination at physiological conditions.

  12. DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs).

    Science.gov (United States)

    Arulandhu, Alfred J; van Dijk, Jeroen P; Dobnik, David; Holst-Jensen, Arne; Shi, Jianxin; Zel, Jana; Kok, Esther J

    2016-07-01

    With the increased global production of different genetically modified (GM) plant varieties, chances increase that unauthorized GM organisms (UGMOs) may enter the food chain. At the same time, the detection of UGMOs is a challenging task because of the limited sequence information that will generally be available. PCR-based methods are available to detect and quantify known UGMOs in specific cases. If this approach is not feasible, DNA enrichment of the unknown adjacent sequences of known GMO elements is one way to detect the presence of UGMOs in a food or feed product. These enrichment approaches are also known as chromosome walking or gene walking (GW). In recent years, enrichment approaches have been coupled with next generation sequencing (NGS) analysis and implemented in, amongst others, the medical and microbiological fields. The present review will provide an overview of these approaches and an evaluation of their applicability in the identification of UGMOs in complex food or feed samples.

  13. Selective detection and quantification of modified DNA with solid-state nanopores.

    Science.gov (United States)

    Carlsen, Autumn T; Zahid, Osama K; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

    2014-10-08

    We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

  14. Targeting human c-Myc promoter duplex DNA with actinomycin D by use of multi-way analysis of quantum-dot-mediated fluorescence resonance energy transfer

    DEFF Research Database (Denmark)

    Gholami, Somayeh; Kompany Zare, Mohsen

    2013-01-01

    was investigated by use of 2D-photoluminescence emission (2D-PLE), and the resulting data were subjected to analysis by use of convenient and powerful multi-way approaches. Fluorescence measurements were performed by use of the quantum dot (QD)-conjugated c-Myc promoter. Intercalation of 7AAD within duplex base...

  15. NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (εdA) opposite thymidine in a DNA duplex. Nonplanar alignment of εdA(anti) and dT(anti) at the lesion site

    International Nuclear Information System (INIS)

    Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Yarema, K.; Basu, A.; Essigmann, J.

    1991-01-01

    Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C)·d(G-T-A-C-εA-C-A-T-G) nonanucleotide duplex (designated εdA·dT 9-mer duplex) containing 1,N 6 -ethenodeoxyadenosine (εdA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. The authors NMR studies have focused on the conformation of the εdA·dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5·εdA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and εdA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4·dC15 and dG6·dC13 pairs. Furthermore, the d(G4-T5-G6)·d(C13-εA14-C15) trinucleotide segment centered about the dT5·εdA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and εdA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the εdA·dT 9-mer duplex. The NMR data are consistent with a nonplanar alignment of εdA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4·dC15 base pair within the d(G4-T5-G6)·d(C13-εA14-C15) segment of the εdA·dT 9-mer duplex

  16. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Science.gov (United States)

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Duplex and Triplex Formation of Mixed Pyrimidine Oligonucleotides with Stacking of Phenyl-triazole Moieties in the Major Groove

    DEFF Research Database (Denmark)

    Andersen, Nicolai Krog; Døssing, Holger; Jensen, Frank

    2011-01-01

    5-(1-Phenyl-1,2,3-triazol-4-yl)-2'-deoxycytidine was synthesized from a modified CuAAC protocol and incorporated into mixed pyrimidine oligonucleotide sequences together with the corresponding 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine. With consecutive incorporations of the two modified...... nucleosides, improved duplex formation with a complementary RNA and improved triplex formation with a complementary DNA duplex were observed. The improvement is due to π-π stacking of the phenyl-triazole moieties in the major groove. The strongest stacking and most pronounced positive influence on thermal...... stability was found in between the uridine analogues or with the cytidine analogue placed in the 3' direction to the uridine analogue. Modeling indicated a different orientation of the phenyl-triazole moieties in the major groove to account for the difference between the two nucleotides. The modified...

  18. Exploring the Role of Genetic Modifiers in DNA Repair and Breast Cancer

    Science.gov (United States)

    2013-09-01

    09-1-0029 TITLE: Exploring the role of genetic modifiers in DNA repair and breast cancer PRINCIPAL INVESTIGATOR: Brian D...1 September 2009 - 31 August 2013Annual SummarySeptember 2013 Exploring the Role of Genetic Modifiers in DNA Repair and Breast Cancer Brian D...cause sensitivity to DNA damage in the yeast Saccharomyces cerevisiae in an attempt to identify genetic factors which cause predisposition to breast

  19. Modifying and adapting a plant-based DNA extraction protocol for ...

    African Journals Online (AJOL)

    ... a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

  20. LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

    Science.gov (United States)

    Maciaszek, Anna; Krakowiak, Agnieszka; Janicka, Magdalena; Tomaszewska-Antczak, Agnieszka; Sobczak, Milena; Mikołajczyk, Barbara; Guga, Piotr

    2015-02-28

    Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

  1. Detecting deletions, insertions, and single nucleotide substitutions in cloned β-globin genes and new polymorphic nucleotide substitutions in β-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA: DNA duplexes

    International Nuclear Information System (INIS)

    Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

    1990-08-01

    The applicability of ribonuclease (RNase) cleavage at mismatches in RNA:DNA duplexes (the RNase cleavage method) for determining nucleotide variant rates was examined in a Japanese population. DNA segments of various lengths obtained from four different regions of one normal and three thalassemic cloned human β-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32 P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of one (G), four(TTCT), five (ATTTT), and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the β-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allene T) was 0.52. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. We conclude that it would be feasible to use the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA. (J.P.N.)

  2. Replacing antibodies with modified DNA aptamers in vaccine potency assays.

    Science.gov (United States)

    Trausch, Jeremiah J; Shank-Retzlaff, Mary; Verch, Thorsten

    2017-10-04

    Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM 197 . Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM 197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM 197 , a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Conformation of amine-modified DNA: 2-aminofluorene- and 2-(acetylamino)fluorene-modified deoxydinucleoside monophosphates with all possible nearest neighbors. A comparison of search and optimization methods.

    Science.gov (United States)

    Shapiro, R; Sidawi, D; Miao, Y S; Hingerty, B E; Schmidt, K E; Moskowitz, J; Broyde, S

    1994-01-01

    Although a significant part of the replication fork exists as single-stranded DNA, little is known about the effect of carcinogens and mutagens on single-strand conformation. Large-scale conformational searches with potential energy minimization, using the torsion angle space molecular mechanics program DUPLEX, were employed to explore the conformation of all 16 deoxydinucleoside monophosphates bearing 2-aminofluorene (AF) or 2-(acetylamino)fluorene (AAF) modification on guanine. We have thus examined the effect of 3' versus 5' modification, the presence or absence of the acetyl group, and the effect of four different neighbors in each case. The principal effect of the acetyl group appeared to be the destabilization of anti (and, to a lesser degree, borderline anti) conformations for modified guanine. This mattered little in the 5'-substituted dimers, where one conformational type predominated in the low-energy structures for the adducts of both AAF and AF: It was right-handed, with syn-guanine, imperfect base-base stacking, and fluorene to 3'-sugar contacts. Greater divergence was seen in the 3'-substituted series. The AAF-substituted 3'-adducts primarily displayed good base-fluorene stacking, with syn-guanine in contact with the 5'-sugar. The AF-substituted 3'-adducts displayed a variety of structures which included base-base and carcinogen-base stacked forms. Two novel forms were encountered [global minima for d(ApG-AF) and d(GpG-AF)], whose unusual structures suggest mutagenic capability. In order to address the multiple minimum problem, we conducted our searches of conformation space using two alternative optimization methods that also employ differing search strategies. We used the Powell algorithm, BOTM, with starting conformations that are selected combinations of rotamers, and the method of simulated annealing (SA), with random or arbitrary starting conformations. While both approaches were effective in defining the most important structures, SA was more

  4. Preferential recognition of auto-antibodies against 4-hydroxynonenal modified DNA in the cancer patients.

    Science.gov (United States)

    Faisal, Mohammad; Shahab, Uzma; Alatar, Abdulrahman A; Ahmad, Saheem

    2017-11-01

    The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes. The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA. DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients. The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients. © 2017 Wiley Periodicals, Inc.

  5. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  6. DNA hybridization on membrane-modified carbon electrodes

    Czech Academy of Sciences Publication Activity Database

    Kouřilová, Alena; Babkina, S. S.; Cahová, Kateřina; Havran, Luděk; Jelen, František; Paleček, Emil; Fojta, Miroslav

    2005-01-01

    Roč. 38, - (2005), s. 2493-2507 ISSN 0003-2719 R&D Projects: GA MPO(CZ) 1H-PK/42; GA AV ČR(CZ) IAA4004402; GA AV ČR(CZ) IBS5004355 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA hybridization * electrochemical DNA sensor * nitrocellulose membrane Subject RIV: BO - Biophysics Impact factor: 1.036, year: 2005

  7. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

  8. Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Hocek, Michal

    2009-01-01

    Roč. 37, č. 22 (2009), s. 7612-7622 ISSN 0305-1048 R&D Projects: GA ČR GA203/09/0317; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : functionalized DNA * restriction endonucleases * DNA polymerase * modified adenosines Subject RIV: CC - Organic Chemistry Impact factor: 7.479, year: 2009

  9. Cleavage of Functionalized DNA Containing 5-Modified Pyrimidines by Type II Restriction Endonucleases

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Pohl, Radek; Hocek, Michal

    2011-01-01

    Roč. 12, č. 3 (2011), s. 431-438 ISSN 1439-4227 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : base-modified DNA * DNA cleavage * DNA polymerases * nucleosides * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 3.944, year: 2011

  10. Measurement and meaning of oxidatively modified DNA lesions in urine

    DEFF Research Database (Denmark)

    Cooke, Marcus S; Olinski, Ryszard; Loft, Steffen

    2008-01-01

    G levels and is not sufficiently specific for accurate quantification. With increasing numbers of lesions being studied, it is vital that these fundamental issues are addressed. We report the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis whose primary goal is to achieve......BACKGROUND: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies....... METHODS: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine. RESULTS: Most of the literature focus upon 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical...

  11. Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II)

    International Nuclear Information System (INIS)

    Sundquist, W.I.; Lippard, S.J.; Stollar, B.D.

    1987-01-01

    Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemothrapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)/sub b/, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations of cis-DDP bound to DNA. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl 2 ], [Pt(dach)Cl 2 ], and [Pt(NH 3 ) 2 (cbdca)] (carboplatin) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA. This antibody crossreacted with unmodified, denatured DNA. The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities

  12. Increased Y-chromosome detection by SRY duplexing

    DEFF Research Database (Denmark)

    Hansen, Morten Høgh; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2012-01-01

    Determining fetal sex noninvasively is dependent of a robust assay. We designed a novel SRY assay and combined it with a SRY assay from literature forming a duplex assay with the same fluorescent dye to increase detection of Y-chromosome at low cell-free fetal DNA or chimeric DNA concentrations....

  13. High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

    Directory of Open Access Journals (Sweden)

    Orada Chumphukam

    2014-04-01

    Full Text Available We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE. One selected aptamer sequence (R15/19 has a high affinity towards the enzyme (Kd = 157 ± 42 pM. Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM, however significant reduction in affinity occurred at high ionic strength (~1.2 M. In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

  14. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    Science.gov (United States)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  15. Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

    International Nuclear Information System (INIS)

    Japoni, A.; Alborzi, A.; Rasouli, M.; Pourabbas, B.

    2004-01-01

    Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of staphylococcus aureus and 50 isolates of coagulase negative staphylococci was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with Mic>8 μ g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

  16. Electrochemical impedance-based DNA sensor using a modified single walled carbon nanotube electrode

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Jessica E. [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Pillai, Shreekumar [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States); Ram, Manoj Kumar, E-mail: mkram@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Kumar, Ashok [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Singh, Shree R. [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States)

    2011-07-20

    Carbon nanotubes have become promising functional materials for the development of advanced electrochemical biosensors with novel features which could promote electron-transfer with various redox active biomolecules. This paper presents the detection of Salmonella enterica serovar Typhimurium using chemically modified single walled carbon nanotubes (SWNTs) with single stranded DNA (ssDNA) on a polished glassy carbon electrode. Hybridization with the corresponding complementary ssDNA has shown a shift in the impedance studies due to a higher charge transfer in ssDNA. The developed biosensor has revealed an excellent specificity for the appropriate targeted DNA strand. The methodologies to prepare and functionalize the electrode could be adopted in the development of DNA hybridization biosensor.

  17. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays...... results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false...... positives in the cloning stimulation assay were less than 5%. This conditions could ensure a good performance of the enzyme preparations for cloning experiments. Finally, we described the use of the radiolabeled [gamma 33P] ATP lambda Hpa II DNA substrate to detect 5'-->3' single stranded DNA dependent...

  18. A highly sensitive and selective sensor based on a graphene-coated carbon paste electrode modified with a computationally designed boron-embedded duplex molecularly imprinted hybrid membrane for the sensing of lamotrigine.

    Science.gov (United States)

    Wang, Hongjuan; Qian, Duo; Xiao, Xilin; Gao, Shuqin; Cheng, Jianlin; He, Bo; Liao, Lifu; Deng, Jian

    2017-08-15

    An innovative electrochemical sensor, based on a carbon paste electrode (CPE) modified with graphene (GR) and a boron-embedded duplex molecularly imprinted hybrid membrane (B-DMIHM), was fabricated for the highly sensitive and selective determination of lamotrigine (LMT). Density functional theory (DFT) was employed to study the interactions between the template and monomers to screen appropriate functional monomers for rational design of the B-DMIHM. The distinct synergic effect of GR and B-DMIHM was evidenced by the positive shift of the reduction peak potential of LMT at B-DMIHM/GR modified CPE (B-DMIHM/GR/CPE) by about 300mV, and the 13-fold amplification of the peak current, compared to a bare carbon paste electrode (CPE). The electrochemical reduction mechanism of lamotrigine was investigated by different voltammetric techniques. It was illustrated that square wave voltammetry (SWV) was more sensitive than different pulse voltammetry (DPV) for the quantitative analysis of LMT. Thereafter, a highly sensitive electroanalytical method for LMT was established by SWV at B-DMIHM/GR/CPE with a good linear relationship from 5.0×10 -8 to 5.0×10 -5 and 5.0×10 -5 to 3.0×10 -4 molL -1 with a lower detection limit (1.52×10 -9 molL -1 ) based on the lower linear range(S/N=3). The practical application of the sensor was demonstrated by determining the concentration of LMT in pharmaceutical and biological samples with good precision (RSD 1.04-4.41%) and acceptable recoveries (92.40-107.0%). Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Enzymatic Incorporation of Modified Purine Nucleotides in DNA.

    Science.gov (United States)

    Abu El Asrar, Rania; Margamuljana, Lia; Abramov, Mikhail; Bande, Omprakash; Agnello, Stefano; Jang, Miyeon; Herdewijn, Piet

    2017-12-14

    A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N 8 -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases.

    Science.gov (United States)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper; Veedu, Rakesh N

    2012-07-15

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Simplex and duplex event-specific analytical methods for functional biotech maize.

    Science.gov (United States)

    Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

    2009-08-26

    Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

  2. Energetics, conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear PtII complex

    Czech Academy of Sciences Publication Activity Database

    Mlčoušková, J.; Malina, Jaroslav; Novohradský, V.; Kašpárková, J.; Komeda, S.; Brabec, Viktor

    2012-01-01

    Roč. 1820, č. 10 (2012), s. 1502-1511 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GAP205/11/0856 Institutional research plan: CEZ:AV0Z50040702 Keywords : DNA * platinum * DNA repair Subject RIV: BO - Biophysics Impact factor: 3.848, year: 2012

  3. Oligopyridine-ruthenium(II)-amino acid conjugates: synthesis, characterization, DNA binding properties and interactions with the oligonucleotide duplex d(5'-CGCGCG-3')2

    Czech Academy of Sciences Publication Activity Database

    Triantafillidi, K.; Karidi, K.; Malina, Jaroslav; Garoufis, A.

    -, č. 32 (2009), s. 6403-6415 ISSN 1477-9226 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : oligopyridine- ruthenium (II)-amino acid conjugates * diastereomeric DNA binding * DNA photocleavage Subject RIV: BO - Biophysics Impact factor: 4.081, year: 2009

  4. Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

    Science.gov (United States)

    Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

    2014-01-01

    DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

  5. Immunological detection of modified DNA bases in irradiated food

    International Nuclear Information System (INIS)

    Williams, J.H.H.; Tyreman, A.L.; Deeble, D.J.; Jones, M.; Smith, C.J.; Christiansen, J.F.; Beaumont, P.C.

    1996-01-01

    Ionising radiation is fatal to all known life forms given sufficient exposure in terms of dose and duration. This property has been used beneficially to sterilise a range of materials, particularly medical products where the removal of all contaminating organisms is deemed essential. Irradiation has long been used to sterilise food for consumption by certain categories of patients. The method is attractive because all potentially contaminating organisms can be removed by one simple treatment. Irradiation also slows down or stops certain processes such as sprouting. There are, however, disadvantages to irradiating food. It is not only DNA that is affected as alterations in lipid and protein components of the food may lead to a loss of quality. Although irradiation will kill bacteria it will probably not affect any toxin produced by those bacteria prior to treatment. Irradiation achieves its effects by damaging molecules, particularly nucleic acids. Consequently, if any of the damage to nucleic acids could be shown to by by a process unique to irradiation, and the products of this unique process could be measured, then there is the basis for a detection system. Furthermore, if the damage could be shown to be proportional to the dose of radiation received then the dose could also be quantified. Legislation, therefore, requires that assays be developed for use in different countries, those which totally ban irradiated food, those which require irradiated food to be labelled and those which have selective laws relating to specific foods and specific levels of irradiation. (author)

  6. Voltammetric Detection of Antioxidative Properties of Flavonoids Using Electrically Heated DNA Modified Carbon Paste Electrode

    Directory of Open Access Journals (Sweden)

    P. Gründler

    2003-01-01

    Full Text Available A simple electrochemical sensor consisting of electrically heated carbon paste electrode with the surface modified by dsDNA is used to characterize voltammetric behaviour and antioxidative activity of four selected flavonoids. Quercetin, rutin, catechin and epigallocatechin gallate accumulate within the DNA layer. A positive effect of the electrode temperature within the range of 20 to 38 ºC on the detection of a deep DNA degradation by a copper(II/H2O2/ascorbic acid cleavage mixture as well as an antioxidative effect of flavonoids was evaluated.

  7. Coarse-grained molecular dynamics simulation of DNA translocation in chemically modified nanopores.

    Science.gov (United States)

    Ramachandran, Abhijit; Guo, Qingjiang; Iqbal, Samir M; Liu, Yaling

    2011-05-19

    Solid-state nanopores provide a direct means to detect and analyze DNA and proteins. In a typical setup, the DNA molecules travel through a nanopore under electrophoretic voltage bias. The nanopore is sandwiched between two chambers that are filled with ionic solution. A major challenge in using solid-state nanopores for DNA sequencing and gene detection is to improve their selectivity and detection sensitivity. To achieve these goals, one solution is to functionalize the nanopores by chemically modifying the pore walls with silanes or nucleic acids. However, little is known about molecular interactions in functionalized nanopores. This paper presents DNA translocation dynamics and the mechanism of DNA sequencing in a functionalized nanopore through a coarse-grained molecular dynamics model. The DNA nucleotide is coarse-grained into two interaction sites: one site corresponds to the base group and the other encompasses the phosphate and sugar groups. The water molecules are included in the model implicitly through Langevin dynamics. The coarse-grained model immensely improves the computational efficiency while still capturing the essential translocation dynamics. The model characterizes important physical properties of functionalized nanopores such as the effective pore diameter and effect of biasing voltage on the DNA translocation dynamics. The model reveals a nonlinear relationship between translocation speed of DNA and applied voltage. Moreover, DNA translocation in nanopores functionalized with hairpin-loop (HPL) DNA and single-stranded DNA (ss-DNA) shows significant differences: a target DNA is found to translocate through a ss-DNA coated nanopore 9 times faster than through an HPL coated one at a bias of 100 mV, putatively from lower stiffness of ss-DNA than that for HPL. The DNA translocation speed is also largely influenced by interaction potential between the DNA and surface-tethered molecules. The results reveal that such selective translocation

  8. Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers

    DEFF Research Database (Denmark)

    Karmakar, Saswata; Madsen, Andreas Stahl; Guenther, Dale C.

    2014-01-01

    Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and more recently engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating......'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA...

  9. Gold nanoparticle-based 2'-O-methyl modified DNA probes for breast cancerous theranostics.

    Science.gov (United States)

    Li, Jing; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Xie, Nuli; Wu, Yanan; Ma, Changbei; Wang, Kemin

    2018-06-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulated diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways. An increasing number of data suggested that miRNA-21 could be identified as diagnostic and therapeutic biomarker for breast cancer. Meanwhile, inhibiting the function of miRNA-21, resulting in cells growth inhibition and apoptotic cells death. To realize miRNA-21detection and inhibition to diagnostic and therapeutic breast cancer cells, we developed gold nanoparticle-based 2'-O-methyl modified DNA probes (AuNP-2'-OMe-DNA probes) for diagnostic and therapeutic breast cancer. Gold nanoparticles were functionalized with chemically modified miRNA-21 inhibitor to suppress the function of miRNA-21 for the therapeutic breast cancer, at the same time, fluorophore-labeled DNA molecules were hybridized with antimiRNA-21 for diagnostic breast cancer. The results showed that the 2'-O-methyl modified DNA can improve stability, increase binding affinity to target strands and enhance the therapeutic effects. The experimental results also demonstrated that antimiR-21 were efficiently introduced into the cells and knocked down miRNA-21 to inhibit its function, leading to growth inhibition and apoptotic cells death. We prospected that chemically modified miRNA-21 inhibitor based on gold nanoparticles would be as a promising diagnostic and therapeutic platform for breast cancer clinically. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Genetic modifiers of carcinogen DNA adducts in target lung and peripheral blood mononuclear cells.

    Science.gov (United States)

    Lee, Mi-Sun; Su, Li; Mark, Eugene J; Wain, John C; Christiani, David C

    2010-12-01

    Measurement of carcinogen DNA adducts in blood has been used as a surrogate for the target lung tissue. We aimed to examine whether genetic polymorphisms in several metabolic pathway genes modify the relation between DNA adducts in target lung and blood. One hundred and thirty-five early-stage lung cancer patients from the Massachusetts General Hospital were studied. DNA adducts were measured by the (32)P-postlabeling assay in lung and blood mononuclear cells (MNCs) in a subset of 53 who had paired blood samples. Single-nucleotide polymorphisms (SNPs) were assessed in genes involved in phase II (GSTs, NAT2, EPHX and NQO1), DNA repair (ERCC1, ERCC2 and XRCC1) and DNA methylation (MTHFR C677T and A1298C) pathways. There was a significant correlation between DNA adduct levels in lung and blood within the different genotypes, with one exception. Significant modifications in adducts were found by variants in genes for phase II metabolism [NAT2 (1.51 for rapid versus 0.76 for slow, P = 0.022)], DNA repair [ERCC1 C118T (P = 0.014), ERCC2 (P = 0.003) and XRCC1 (P = 0.025)] and MTHFR [C677T (P = 0.005) and A1298C (P = 0.005)]. The relation between DNA adducts in blood MNCs and target lung tissue was significantly modified by the single-nucleotide polymorphisms in the three main pathways. Despite the relatively small sample size, our results suggest that genetic factors may need to be considered when assessing the association of DNA adducts using surrogate tissue in studies of lung cancer. Further studies are needed to better understand their role and the mechanisms.

  11. Synthesis, hybridization and fluorescence properties of a 2'-C-pyrene-triazole modified arabino-uridine nucleotide

    DEFF Research Database (Denmark)

    Kumar, Pawan; Sharma, Pawan K.; Nielsen, Poul

    2017-01-01

    A new pyrene-modified nucleotide monomer is introduced, wherein pyrene is attached to the 2'-position of arabino-uridine through a triazolemethyl linker. When the monomer is introduced into oligonucleotides, very stable DNA duplexes and three way junctions are obtained. An oligonucleotide featuri...

  12. Directed Formation of DNA Nanoarrays through Orthogonal Self-Assembly

    Directory of Open Access Journals (Sweden)

    Eugen Stulz

    2011-06-01

    Full Text Available We describe the synthesis of terpyridine modified DNA strands which selectively form DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. The short DNA strands are designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as Zn(II and Ni(II induces the formation of tubular arrays consisting of DNA bundles which are 50-200 nm wide and 2-50 nm high. TEM shows additional long distance ordering of the terpy-DNA complexes into fibers.

  13. Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

    Science.gov (United States)

    Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

    2012-07-01

    The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

  14. Voltammetry of osmium-modified DNA at a mercury film electrode application in detecting DNA hybridization

    Czech Academy of Sciences Publication Activity Database

    Kostečka, Pavel; Havran, Luděk; Pivoňková, Hana; Fojta, Miroslav

    2004-01-01

    Roč. 63, 1-2 (2004), s. 245-248 ISSN 1567-5394 R&D Projects: GA AV ČR IAA4004108; GA AV ČR KJB4004302 Institutional research plan: CEZ:AV0Z5004920 Keywords : osmium * DNA hybridization * mercury film electrode Subject RIV: BO - Biophysics Impact factor: 2.261, year: 2004

  15. Electrooxidation of DNA at glassy carbon electrodes modified with multiwall carbon nanotubes dispersed in polyethylenimine

    Energy Technology Data Exchange (ETDEWEB)

    Luque, Guillermina L.; Ferreyra, Nancy F. [INFIQC, Departamento de Fisico Quimica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina); Granero, Adrian [INFIQC, Departamento de Fisico Quimica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina); Departamento de Quimica, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Rio Cuarto (Argentina); Bollo, Soledad [Laboratorio de Bioelectroquimica, Facultad de Ciencias Quimicas y Farmaceuticas, Universidad de Chile, P.O. Box 233, Santiago (Chile); Rivas, Gustavo A., E-mail: grivas@fcq.unc.edu.ar [INFIQC, Departamento de Fisico Quimica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-10-30

    This work reports the electrochemical response of the complex between dsDNA and PEI formed in solution and at the surface of glassy carbon electrodes (GCE) modified with a dispersion of multi-walled carbon nanotubes in polyethylenimine (CNT-PEI). Scanning Electron Microscopy and Scanning Electrochemical Microscopy demonstrate that the dispersion covers the whole surface of the electrode although there are areas with higher density of CNT and, consequently, with higher electrochemical reactivity. The adsorption of DNA at GCE/CNT-PEI is fast and it is mainly driven by electrostatic forces. A clear oxidation signal is obtained either for dsDNA or a heterooligonucleotide of 21 bases (oligoY) at potentials smaller than those for the oxidation at bare GCE. The comparison of the behavior of DNA before and after thermal treatment demonstrated that the electrochemical response highly depends on the 3D structure of the nucleic acid.

  16. High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    DEFF Research Database (Denmark)

    Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

    2009-01-01

    length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally......While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine...... substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na(+)). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer...

  17. Electrooxidation of DNA at glassy carbon electrodes modified with multiwall carbon nanotubes dispersed in polyethylenimine

    International Nuclear Information System (INIS)

    Luque, Guillermina L.; Ferreyra, Nancy F.; Granero, Adrian; Bollo, Soledad; Rivas, Gustavo A.

    2011-01-01

    This work reports the electrochemical response of the complex between dsDNA and PEI formed in solution and at the surface of glassy carbon electrodes (GCE) modified with a dispersion of multi-walled carbon nanotubes in polyethylenimine (CNT-PEI). Scanning Electron Microscopy and Scanning Electrochemical Microscopy demonstrate that the dispersion covers the whole surface of the electrode although there are areas with higher density of CNT and, consequently, with higher electrochemical reactivity. The adsorption of DNA at GCE/CNT-PEI is fast and it is mainly driven by electrostatic forces. A clear oxidation signal is obtained either for dsDNA or a heterooligonucleotide of 21 bases (oligoY) at potentials smaller than those for the oxidation at bare GCE. The comparison of the behavior of DNA before and after thermal treatment demonstrated that the electrochemical response highly depends on the 3D structure of the nucleic acid.

  18. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays...... exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I....... results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false...

  19. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Liu Lanxia; Bai Yuanyuan; Song Chunni; Zhu Dunwan; Song Liping; Zhang Hailing; Dong Xia; Leng Xigang, E-mail: lengxg@bme.org.c [Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Laboratory of Bioengineering (China)

    2010-06-15

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-10, IL-12, and TNF-{alpha}). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 {mu}g/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  20. Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

    Science.gov (United States)

    Hocek, Michal

    2014-11-07

    The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

  1. A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

    Energy Technology Data Exchange (ETDEWEB)

    Oskuee, Reza Kazemi [Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Dosti, Fatemeh [School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Gholami, Leila [Targeted Drug Delivery Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Malaekeh-Nikouei, Bizhan, E-mail: malaekehb@mums.ac.ir [Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

    2015-04-01

    Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

  2. A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

    International Nuclear Information System (INIS)

    Oskuee, Reza Kazemi; Dosti, Fatemeh; Gholami, Leila; Malaekeh-Nikouei, Bizhan

    2015-01-01

    Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

  3. DNA binding and cleavage studies of copper(II) complexes with 2'-deoxyadenosine modified histidine moiety.

    Science.gov (United States)

    Borowska, Justyna; Sierant, Malgorzata; Sochacka, Elzbieta; Sanna, Daniele; Lodyga-Chruscinska, Elzbieta

    2015-09-01

    This work is focused on the study of DNA binding and cleavage properties of 2'-deoxyadenosines modified with ester/amide of histidine (his(6)dA ester, his(6)dA amide) and their copper(II) complexes. To determine the coordination mode of the complex species potentiometric and spectroscopic (UV-visible, CD, EPR) studies have been performed. The analysis of electronic absorption and fluorescence spectra has been used to find the nature of the interactions between the compounds and calf thymus DNA (CT-DNA). There is significant influence of the -NH2 and -OCH3 groups on binding of the ligands or the complexes to DNA. Only amide derivative and its complex reveal intercalative ability. In the case of his(6)dA ester and Cu(II)-his(6)dA ester the main interactions can be groove binding. DNA cleavage activities of the compounds have been examined by gel electrophoresis. The copper complexes have promoted the cleavage of plasmid DNA, but none of the ligands exhibited any chemical nuclease activity. The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.

  4. Energy band gap and optical transition of metal ion modified double crossover DNA lattices.

    Science.gov (United States)

    Dugasani, Sreekantha Reddy; Ha, Taewoo; Gnapareddy, Bramaramba; Choi, Kyujin; Lee, Junwye; Kim, Byeonghoon; Kim, Jae Hoon; Park, Sung Ha

    2014-10-22

    We report on the energy band gap and optical transition of a series of divalent metal ion (Cu(2+), Ni(2+), Zn(2+), and Co(2+)) modified DNA (M-DNA) double crossover (DX) lattices fabricated on fused silica by the substrate-assisted growth (SAG) method. We demonstrate how the degree of coverage of the DX lattices is influenced by the DX monomer concentration and also analyze the band gaps of the M-DNA lattices. The energy band gap of the M-DNA, between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), ranges from 4.67 to 4.98 eV as judged by optical transitions. Relative to the band gap of a pristine DNA molecule (4.69 eV), the band gap of the M-DNA lattices increases with metal ion doping up to a critical concentration and then decreases with further doping. Interestingly, except for the case of Ni(2+), the onset of the second absorption band shifts to a lower energy until a critical concentration and then shifts to a higher energy with further increasing the metal ion concentration, which is consistent with the evolution of electrical transport characteristics. Our results show that controllable metal ion doping is an effective method to tune the band gap energy of DNA-based nanostructures.

  5. 2′-O-methyl nucleotide modified DNA substrates influence the ...

    Indian Academy of Sciences (India)

    2014-07-18

    Jul 18, 2014 ... enzymatic cleavage efficiency for applications in synthetic biology. [Tong Z, Zhao B, Zhao G, Shang H and Guan Y 2014 2′-O-methyl-nucleotide-modified DNA substrates influence the cleavage efficiencies of. BamHI and BglII. J. Biosci. 39 621–630] DOI 10.1007/s12038-014-9466-4. 1. Introduction. Due to ...

  6. Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines

    Czech Academy of Sciences Publication Activity Database

    Pivoňková, Hana; Horáková Brázdilová, Petra; Fojtová, Miloslava; Fojta, Miroslav

    2010-01-01

    Roč. 82, č. 16 (2010), s. 6807-6813 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : modified DNA * 7-deazapurines * voltammetric analysis Subject RIV: BO - Biophysics Impact factor: 5.874, year: 2010

  7. Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

    Science.gov (United States)

    Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

    2017-12-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

    KAUST Repository

    Wang, Zhijuan

    2011-05-01

    Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

  9. Isolation/separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent.

    Science.gov (United States)

    Chen, Xu-Wei; Mao, Quan-Xing; Liu, Jia-Wei; Wang, Jian-Hua

    2012-10-15

    Hemoglobin (Hb) modified magnetic nanocomposites are prepared by immobilization of Hb onto the surface of amino-functionalized Fe(3)O(4)@SiO(2) magnetic nanoparticles via covalent bonding with glutaraldehyde as cross-linker. The obtained nanocomposites are characterized with FT-IR, SEM, XRD and surface charge analysis. A direct solid-phase extraction procedure for the isolation/separation of plasmid DNA using this nanocomposite as a novel adsorbent is thus developed. Some important experimental parameters governing the sorption efficiency, i.e., the pH of sample solution and the ionic strength, are investigated. The Hb modified magnetic nanocomposites provide a sorption capacity of 27.86 mg g(-1) for DNA. By using 2.0mg of the nanocomposites as sorption medium and a suitable acidity of pH 6.1, a sorption efficiency of 93% is achieved for 25 μg mL(-1) of DNA in 1.0 mL of sample solution. Afterwards, the absorbed DNA could be readily recovered by using 1.0 mL of Tris-HCl buffer (pH 8.9, 0.01 mol L(-1)), giving rise to a recovery of ca. 68.3%. The present solid-phased extraction protocol is applied for the isolation of plasmid DNA from Escherichia coli culture, resulting in comparable yield and purity of plasmid DNA with respect to those obtained by using commercial kits. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2008-06-01

    Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

  11. Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

    Science.gov (United States)

    Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

    2018-04-01

    DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

  12. AFFINITY BIOSENSOR BASED ON SCREEN-PRINTED ELECTRODE MODIFIED WITH DNA FOR GENOTOXIC COMPOUNDS DETECTION

    Directory of Open Access Journals (Sweden)

    Bambang Kuswandi

    2010-06-01

    Full Text Available An electrochemical method for the detection of the genotoxic compounds using a DNA-modified electrode was developed. This electrode was successfully used for the electrochemical detection of genotoxic compounds in water samples. The electrochemical results clearly demonstrated that, the development is related to the molecular interaction between the surface-linked DNA obtained from calf thymus and the target compounds, such as pollutants, in order to develop a simple device for rapid screening of genotoxic compounds in environmental samples. The detection of such compounds was measured by their effect on the oxidation signal of the guanine peak of the DNA immobilised on the surface of carbon based Screen-Printed Electrode (SPE in disposable mode, and monitored by square-wave voltametric analysis. The DNA biosensor is able to detect known intercalating and groove-binding genotoxic compounds such as Dioxin, Bisphenol A, PCBs, and Phtalates. Application to real water samples is discussed and reported.   Keywords: electrochemical, screen-printed electrode, DNA biosensor, genotoxic compounds

  13. Detection of genetically modified crops by combination of multiplex PCR and low-density DNA microarray.

    Science.gov (United States)

    Zhou, Ping-Ping; Zhang, Jian-Zhong; You, Yuan-Hai; Wu, Yong-Ning

    2008-02-01

    To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1). A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

  14. Immunological detection and quantification of DNA components structurally modified by alkylating carcinogens, mutagens and chemotherapeutic agents

    International Nuclear Information System (INIS)

    Rajewsky, M.F.

    1983-01-01

    The detection and quantification of defined reaction products of chemical mutagens and carcinogens (and of many cancer chemotherapeutic agents) with DNA require highly sensitive analytical techniques. The exceptional capability of immunoglobulins to recognize subtle alterations of molecular structure (especially when monoclonal antibodies are used to maximize specificity), outstanding sensitivity of immunoanalysis by high-affinity antibodies, and the fact that radioactively-labelled agents are not required suggest the utility of a radioimmunoassay to recognize and quantitate alkylated DNA products. We have recently developed a set of high-affinity monoclonal antibodies (secreted by mouse x mouse as well as by rat x rat hybridomas; antibody affinity constants, 10 9 to > 10 10 lmol) specifically directed against several DNA alkylation products with possible relevance in relation to both mutagenesis and malignant transformation of mammalian cells. These alkylation products include 0 6 -N-butyldeoxyguanosine, and 0 4 -ethyldeoxythymidine. When used in a radioimmunassay, an antibody specific for 0 6 -ethyldeoxyguanosine, for example, will detect this product at an 0 6 -ethyldeoxyguanosine/deoxyguanosine molar ratio of approx. 3 x 10 -7 in a hydrolysate of 100 ug of DNA. The limit of detection can be lowered further if the respective alkyldeoxynucleosides are separated by HPLC from the DNA hydrolysate prior to the RIA. The anti-alkyldeoxynucleoside monoclonal antibodies can also be used to visualize, by immunostaining and fluorescence microscopy combined with electronic image intensification, specific alkylation products in the nuclear DNA of individual cells, and to localize structurally modified bases in double-stranded DNA molecules by transmission electron microscopy

  15. Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

    Science.gov (United States)

    Samson, Maria Cristina; Gullì, Mariolina; Marmiroli, Nelson

    2010-07-01

    Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

  16. Efficient energy transfer between coronene-modified permethyl-β-cyclodextrins and porphyrin for light induced DNA cleavage.

    Science.gov (United States)

    Yu, Jie; Zhang, Ying-Ming; Li, Pei-Yu; Liu, Yu

    2017-03-28

    A novel supramolecular assembly was constructed by the noncovalent complexation of hexa-cata-hexabenzocoronene modified permethyl-β-cyclodextrins with tetrasodium tetraphenylporphyrintetrasulfonate in water, exhibiting highly efficient excited energy transfer behaviors and a promising DNA photocleavage ability.

  17. Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy.

    Science.gov (United States)

    Un, Keita; Kawakami, Shigeru; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2010-10-01

    Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

    Science.gov (United States)

    Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

    2017-11-01

    5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A general method to modify BACs to generate large recombinant DNA fragments.

    Science.gov (United States)

    Shen, Wei; Huang, Yue; Tang, Yi; Liu, De-Pei; Liang, Chih-Chuan

    2005-11-01

    Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

  20. Welding of duplex and super-duplex stainless steels

    International Nuclear Information System (INIS)

    Van Nassau, L.; Meelker, H.; Hilkes, J.

    1994-01-01

    After a recall of the commercial designation of duplex or super-duplex steels (22-27% Cr, 4-8% Ni, 0.1-0.3% N with or without Mo (1.5-4%)) and of some metallurgical properties (phase diagrams, microstructure, ferrite determination, heat treatment and aging), welding technologies are synthetically presented (advantages-disadvantages of each process, metals filler, parameters of the welding processes, heat treatments after welding, cleaning, passivation, properties (mechanical, corrosion resistance) of the welded pieces). (A.B.). 28 refs. 5 figs., 15 tabs., 1 annexe

  1. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    Science.gov (United States)

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-02

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

  2. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2016-11-15

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  3. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    International Nuclear Information System (INIS)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-01-01

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  4. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  5. Nanoparticle-based DNA biosensor for visual detection of genetically modified organisms.

    Science.gov (United States)

    Kalogianni, Despina P; Koraki, Theodora; Christopoulos, Theodore K; Ioannou, Penelope C

    2006-01-15

    Although screening of raw ingredients and food products for genetically modified organisms (GMO) may be accomplished by detecting either the exogenous DNA or the novel protein, DNA is the preferred analyte because of its superior stability during food processing. The development of DNA biosensors is of increasing importance due to the growing demand for rapid and reliable methods for GMO detection. We report the first DNA biosensor in a dry-reagent dipstick configuration for visual detection and confirmation of GMO-related sequences by hybridization within minutes. The sensor is disposable and does not require special instrumentation. It detects the 35S promoter and nopaline synthase (NOS) terminator sequences that are present in the majority of transgenic plants. The target sequences are amplified by the polymerase chain reaction (PCR) and hybridized (7min) with probes bearing oligo(dA) tail. The biotinylated product is applied to the sensor followed by immersion in the appropriate buffer. Migration of the buffer rehydrates gold nanoparticles conjugated to oligo(dT), which hybridize with the oligo(dA) tails. The hybrids are captured by immobilized streptavidin at the test zone of the sensor giving a characteristic red line due to the accumulation of the nanoparticles. The excess of nanoparticle conjugates are captured at the control zone by immobilized oligo(dA) strands. Amplified 35S or NOS DNA is detectable at 0.16nM. Soybean powder certified reference material with 0.1% GMO content is clearly detectable after 35 and 40 amplification cycles for 35S and NOS sequence, respectively. The sensor was also applied to real samples from various sources.

  6. [Extraction of sperm DNA from mixed stain by the modified differential lysis method combined with silicon bead method].

    Science.gov (United States)

    Han, Hai-Jun; Zhang, Yu-Hong; Yang, Min; Yi, Hai; Yang, Geng-Ye; Jia, Dong-Tao; Lu, Da-Ru

    2014-02-01

    To extract sperm DNA from mixed stain by the modified differential lysis method combined with silicon bead method and to evaluate its application value. Fifty-two mixed stains containing female STR genotypes detected by differential lysis method were collected. The sperm DNA was extracted by the modified method combined with silicon bead method, then genotyped with the Identifiler Kit, and compared with the results of genotyping by the conventional differential lysis method as control. Of the 52 samples, 38 samples with sole male STR genotypes in all loci were detected. The detection rate of male STR genotypes was 98.08% through the modified method combined with silicon bead method. The modified differential lysis method combined with silicon bead method can be used in extraction of sperm DNA from mixed stain.

  7. Identification of genetically modified DNA found in Roundup Ready soybean using gold nanoparticles

    International Nuclear Information System (INIS)

    Jang, Huisoo; Kwak, Cheol Hwan; Kim, Gibum; Kim, Sun Min; Huh, Yun Suk; Jeon, Tae-Joon

    2016-01-01

    The authors describe an SPR sensor chip coated with gold nanoparticles (AuNPs) that enables highly sensitive determination of genetically modified (GM) crops. Detection is based on localized surface plasmon resonance (LSPR) with its known sensitivity to even minute changes in refractive index. The device consists of a halogen light source, a light detector, and a cuvette cell that contains a sensor chip coated with AuNPs. It is operated in the transmission mode of the optical path to enhance the plasmonic signal. The sample solution containing target DNA (e.g. from the GM crop) is introduced into the cuvette with the sensor chip whose surface was functionalized with a capture DNA. Following a 30-min hybridization, the changes of the signal are recorded at 540 nm. The chip responds to target DNA in the 1 to 100 nM concentration range and has a 1 nM detection limit. Features of this sensor chip include a short reaction time, ease of handling, and portability, and this enables on-site detection and in-situ testing. (author)

  8. Stwl modifies chromatin compaction and is required to maintain DNA integrity in the presence of perturbed DNA replication

    NARCIS (Netherlands)

    Yi, X.; Vries, de H.I.; Siudeja, K.; Rana, A.; Lemstra, W.; Brunsting, J.F.; Kok, R.J.M.; Smulders, Y.M.; Schaefer, M.; Dijk, F.; Shang, Y.F.; Eggen, B.J.L.; Kampinga, H.H.; Sibon, O.C.M.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent

  9. Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

    NARCIS (Netherlands)

    Yi, Xia; Vries, Hilda I. de; Siudeja, Katarzyna; Rana, Anil; Lemstra, Willy; Brunsting, Jeanette F.; Kok, Rob M.; Smulders, Yvo M.; Schaefer, Matthias; Dijk, Freark; Shang, Yongfeng; Eggen, Bart J.L.; Kampinga, Harm H.; Sibon, Ody C.M.

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent

  10. Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence.

    Science.gov (United States)

    Paner, T M; Riccelli, P V; Owczarzy, R; Benight, A S

    1996-12-01

    Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5'-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3' linked on both ends by single-strand loops of A, or C, sequences (iota = 2, 3, 4, 6, 8, 10, 14). T sequences (iota = 2, 3, 4, 6, 8, 10), and G iota sequences (iota = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na+. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation delta Gloop(n > iota) and single-strand circles, delta Gcir(N) as a function of end-loop size, tau = 2-14, circle size, N = 32 + 2 iota, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy delta H, entropy delta S, and free energy delta G, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na+]. Since the multistate analysis

  11. Achieving single channel, full duplex wireless communication

    KAUST Repository

    Choi, Jung Il

    2010-01-01

    This paper discusses the design of a single channel full-duplex wireless transceiver. The design uses a combination of RF and baseband techniques to achieve full-duplexing with minimal effect on link reliability. Experiments on real nodes show the full-duplex prototype achieves median performance that is within 8% of an ideal full-duplexing system. This paper presents Antenna Cancellation, a novel technique for self-interference cancellation. In conjunction with existing RF interference cancellation and digital baseband interference cancellation, antenna cancellation achieves the amount of self-interference cancellation required for full-duplex operation. The paper also discusses potential MAC and network gains with full-duplexing. It suggests ways in which a full-duplex system can solve some important problems with existing wireless systems including hidden terminals, loss of throughput due to congestion, and large end-to-end delays. Copyright 2010 ACM.

  12. A plasma modified cellulose-chitosan porous membrane allows efficient DNA binding and provides antibacterial properties: A step towards developing a new DNA collecting card.

    Science.gov (United States)

    Chumwangwapee, Sasiwimon; Chingsungnoen, Artit; Siri, Sineenat

    2016-11-01

    In forensic DNA analyses, biological specimens are collected and stored for subsequent recovery and analysis of DNA. A cost-effective and efficient DNA recovery approach is therefore a need. This study aims to produce a plasma modified cellulose-chitosan membrane (pCE-CS) that efficiently binds and retains DNA as a potential DNA collecting card. The pCE-CS membrane was produced by a phase separation of ionic liquid dissolving CE and CS in water with subsequent surface-modification by a two-step exposure of argon plasma and nitrogen gas. Through plasma modification, the pCE-CS membrane demonstrated better DNA retention after a washing process and higher rate of DNA recovery as compared with the original CE-CS membrane and the commercial FTA card. In addition, the pCE-CS membrane exhibited anti-bacterial properties against both Escherichia coli and Staphylococcus aureus. The results of this work suggest a potential function of the pCE-CS membrane as a DNA collecting card with a high recovery rate of captured DNA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2'-Amino-α-l-LNA Adenine Monomers

    DEFF Research Database (Denmark)

    Andersen, Nicolai K; Anderson, Brooke A; Wengel, Jesper

    2013-01-01

    thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides....... ONs modified with pyrene-functionalized 2'-amino-α-l-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation....... These characteristics render N2'-pyrene-functionalized 2'-amino-α-l-LNAs of considerable interest for DNA-targeting applications....

  14. Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

    DEFF Research Database (Denmark)

    Kristoffersen, Emil Laust; Gonzales, Maria; Stougaard, Magnus

    2015-01-01

    Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DN...

  15. CLINICAL-EVALUATION OF A MODIFIED ELISA, USING PHOTOBIOTINYLATED DNA, FOR THE DETECTION OF ANTI-DNA ANTIBODIES

    NARCIS (Netherlands)

    HYLKEMA, MN; HUYGEN, H; KRAMERS, C; VANDERWAL, TJ; DEJONG, J; VANBRUGGEN, MCJ; SWAAK, AJG; BERDEN, JHM; SMEENK, RJT; Hylkema, Machteld

    1994-01-01

    The measurement of anti-dsDNA antibodies is important for the diagnosis and the follow-up of patients with systemic lupus erythematosus (SLE). For routine detection of anti-dsDNA, the Farr assay and the immunofluorescence technique (IFT) on Crithidia luciliae proved to be very useful. The anti-dsDNA

  16. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

    NARCIS (Netherlands)

    Statham, A.L.; Robinson, M.D.; Song, J.Z.; Coolen, M.W.; Stirzaker, C.; Clark, S. J.

    2012-01-01

    The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq),

  17. Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

    Science.gov (United States)

    Manzanares-Palenzuela, C Lorena; de-los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

    2015-06-15

    Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Mitochondrial respiratory modifiers confer survival advantage by facilitating DNA repair in cancer cells

    International Nuclear Information System (INIS)

    Chauhan, Ankit; Khanna, Suchit; Singh, Saurabh; Rai, Yogesh; Soni, Ravi; Kalra, Namita; Dwarakanath, B.S.; Bhatt, Anant Narayan

    2014-01-01

    High rate of aerobic glycolysis (Warburg effect), one of the primary hallmarks of cancer cells, acquired during the multistep development of tumors is also responsible for therapeutic resistance. Underlying this hallmark is the compromised respiratory metabolism that contributes to the acquisition of the glycolytic phenotype for sustained ATP production and cell proliferation. Nevertheless, the exact mechanisms underlying the glycolysis-linked radio-resistance in cancer cells remain elusive. In this study, we transiently elevated glycolysis by treating human cell lines (HEK293, BMG-1 and OCT-1) with mitochondrial respiratory modifiers (MRMs) viz. 2,4-dinitrophenol, Photosan-3, and Methylene blue to examine if transient stimulation of glycolysis before irradiation using MRMs is sufficient to confer radioresistance. Treatment with MRMs led to a significant (two-fold) increase in glucose consumption and lactate production together with a robust increase in the protein levels of two key regulators of glucose metabolism, i.e. GLUT-1 and HK-II. MRMs also enhanced the clonogenic survival and facilitated DNA repair by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. Inhibition of glucose uptake by inhibitors like 2-deoxy-D-glucose (2-DG), 3-bromo pyruvate (3-BP) and fasentin under conditions of stimulated glycolysis not only reversed the effect but also sensitized the cells to radiation more profoundly. The inhibition of glycolysis using 2-DG also reduced the levels of Ku 70 (NHEJ) and Rad-51 (HR) proteins. Thus, our results suggest that enhanced glycolysis in cancer cells may confer radio-resistance and offers survival advantage partly by enhancing the repair of DNA damage. (author)

  19. DNA Computing Systems Activated by Electrochemically-triggered DNA Release from a Polymer-brush-modified Electrode Array

    Science.gov (United States)

    Gamella, Maria; Zakharchenko, Andrey; Guz, Nataliia; Masi, Madeline; Minko, Sergiy; Kolpashchikov, Dmitry M.; Iken, Heiko; Poghossian, Arshak; Schöning, Michael J.; Katz, Evgeny

    2017-01-01

    An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA-based Identity, AND and XOR logic gates. Single-stranded DNA molecules were loaded on the mixed poly(N,N-di-methylaminoethyl methacrylate) (PDMAEMA)/poly-(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAE-MA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals. PMID:29379265

  20. Overall performance of the duplex Stirling refrigerator

    International Nuclear Information System (INIS)

    Erbay, L. Berrin; Ozturk, M. Mete; Doğan, Bahadır

    2017-01-01

    Highlights: • Overall performance coefficient of duplex Stirling refrigerator was investigated. • A definite region for the coefficient of performance of the refrigerator in duplex Stirling is identified. • A definite region for the thermal efficiency of the heat engine in duplex Stirling is identified. • Benchmark values and design bounds of the duplex Stirling refrigerator were obtained. - Abstract: The duplex Stirling refrigerator is an integrated refrigerator consists of Stirling cycle engine and Stirling cycle refrigerator used for cooling. The equality of the work generation of the heat engine to the work consumption of the refrigerator is the primary constraint of the duplex Stirling. The duplex Stirling refrigerator is investigated thermodynamically by considering the effects of constructional and operational parameters which are namely the temperature ratios for heat engine and refrigerator, and the compression ratios for both sides. The primary concern is given to the parametric effects on the overall coefficient of performance of the duplex Stirling refrigerator. The given diagrams provide a design bounds and benchmark results that allows seeing the big picture about the cooling load and heat input relation. Moreover they ease to determine the corresponding work rate to the target cooling load. As regard to the obtained results, a definite region for coefficient of performance of the refrigerator and a definite region for the thermal efficiency of the heat engine of the duplex Stirling are identified.

  1. Fish DNA-modified clays: Towards highly flame retardant polymer nanocomposite with improved interfacial and mechanical performance

    Science.gov (United States)

    Zabihi, Omid; Ahmadi, Mojtaba; Khayyam, Hamid; Naebe, Minoo

    2016-12-01

    Deoxyribonucleic Acid (DNA) has been recently found to be an efficient renewable and environmentally-friendly flame retardant. In this work, for the first time, we have used waste DNA from fishing industry to modify clay structure in order to increase the clay interactions with epoxy resin and take benefit of its additional thermal property effect on thermo-physical properties of epoxy-clay nanocomposites. Intercalation of DNA within the clay layers was accomplished in a one-step approach confirmed by FT-IR, XPS, TGA, and XRD analyses, indicating that d-space of clay layers was expanded from ~1.2 nm for pristine clay to ~1.9 nm for clay modified with DNA (d-clay). Compared to epoxy nanocomposite containing 2.5%wt of Nanomer I.28E organoclay (m-clay), it was found that at 2.5%wt d-clay loading, significant enhancements of ~14%, ~6% and ~26% in tensile strength, tensile modulus, and fracture toughness of epoxy nanocomposite can be achieved, respectively. Effect of DNA as clay modifier on thermal performance of epoxy nanocomposite containing 2.5%wt d-clay was evaluated using TGA and cone calorimetry analysis, revealing significant decreases of ~4000 kJ/m2 and ~78 kW/m2 in total heat release and peak of heat release rate, respectively, in comparison to that containing 2.5%wt of m-clay.

  2. A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations.

    Science.gov (United States)

    Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles

    2015-02-17

    Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a

  3. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

    Science.gov (United States)

    Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

    2014-10-01

    Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

  4. S-adenosylmethionine modifies cocaine-induced DNA methylation and increases locomotor sensitization in mice.

    Science.gov (United States)

    Anier, Kaili; Zharkovsky, Alexander; Kalda, Anti

    2013-10-01

    Several studies suggest that individual variability is a critical component underlying drug addiction as not all members of a population who use addictive substance become addicted. There is evidence that the overall epigenetic status of a cell (epigenome) can be modulated by a variety of environmental factors, such as nutrients and chemicals. Based on these data, our aim was to investigate whether environmental factors like S-adenosylmethionine (SAM) via affecting epigenome could alter cocaine-induced gene expression and locomotor sensitization in mice. Our results demonstrate that repeated SAM (10 mm/kg) pretreatment significantly potentiated cocaine-induced locomotor sensitization. Using mouse nucleus accumbens (NAc) tissue, whole-genome gene expression profiling revealed that repeated SAM treatment affected a limited number of genes, but significantly modified cocaine-induced gene expression by blunting non-specifically the cocaine response. At the gene level, we discovered that SAM modulated cocaine-induced DNA methylation by inhibiting both promoter-associated CpG-island hyper- and hypomethylation in the NAc but not in the reference tissue cerebellum. Finally, our in vitro and in vivo data show that the modulating effect of SAM is in part due to decreased methyltransferase activity via down-regulation of Dnmt3a mRNA. Taken together, our results suggest that environmental factors that affect the NAc-cell epigenome may alter the development of psychostimulant-induced addiction and this may explain, at least partly, why some individuals are more vulnerable to drug addiction.

  5. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  6. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    Science.gov (United States)

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

    Science.gov (United States)

    Halter, Mathew C; Zahn, James A

    2017-02-01

    White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

  8. Role of minor groove width and hydration pattern on amsacrine interaction with DNA.

    Directory of Open Access Journals (Sweden)

    Deepak K Jangir

    Full Text Available Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.

  9. Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

    Directory of Open Access Journals (Sweden)

    Ma G

    2013-03-01

    Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

  10. Control of DNA hybridization by photoswitchable molecular glue.

    Science.gov (United States)

    Dohno, Chikara; Nakatani, Kazuhiko

    2011-12-01

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

  11. The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor

    International Nuclear Information System (INIS)

    Rashid, Jahwarhar Izuan Abdul; Yusof, Nor Azah; Abdullah, Jaafar; Hashim, Uda; Hajian, Reza

    2014-01-01

    This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0–178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4 °C in silica gel. - Highlights: • A sensitive biosensor is presented for detection of dengue virus. • SiNWs and AuNPs used as nanocomposite layers on ITO for construction of biosensor • The detection mechanism is based on the interaction of MB with DNA bonded on AuNPs. • The reduction signal of MB decreases upon complementary hybridization

  12. The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor

    Energy Technology Data Exchange (ETDEWEB)

    Rashid, Jahwarhar Izuan Abdul [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry and Biology, Centre for Defense Foundation Studies, National Defense University of Malaysia, Sungai Besi Camp, 57000 Kuala Lumpur (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Yusof, Nor Azah, E-mail: azahy@upm.edu.my [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Jaafar [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Hashim, Uda [Institute of Nanoelectronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis (Malaysia); Hajian, Reza, E-mail: rezahajian@upm.edu.my [Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

    2014-12-01

    This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0–178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4 °C in silica gel. - Highlights: • A sensitive biosensor is presented for detection of dengue virus. • SiNWs and AuNPs used as nanocomposite layers on ITO for construction of biosensor • The detection mechanism is based on the interaction of MB with DNA bonded on AuNPs. • The reduction signal of MB decreases upon complementary hybridization.

  13. DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

    Science.gov (United States)

    Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

    2015-08-01

    In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products.

  14. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

    Science.gov (United States)

    Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

    2012-01-01

    PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

  15. Conductivity of Pedot-Pss with Gold and Silver Nanocomposites Modified Gold Electrodes for Ganoderma Boninense DNA Detection

    Directory of Open Access Journals (Sweden)

    Sabo Wada Dutse

    2015-08-01

    Full Text Available The conductivity of a designed electrochemical DNA biosensor was improved using gold and or silver nanoparticles. A gold electrode modified with a conductive nanocomposite of poly(3,4-ethylene dioxythiophen–poly (styrenesulfonate (Pedot-Pss and gold or silver nano particles enhanced the conductivity of the electrode surface area. Bare and modified gold electrode surfaces were characterized using cyclic voltammetry (CV technique in ethylenediaminetetraacetic acid (TE supporting electrolyte. Immobilization of a 20-mer DNA probe was achieved by covalent attachment of the amine group of the capture probe to a carboxylic group of an activated 3,3’-dithiodipropionic acid layer using EDC/NHSS for Hybridization. The effect of hybridization temperature and time was optimized and the sensor demonstrated specific detection for the target concentration ranged between 1.0´10-15 M to 1.0´10-9 M with a detection limit of 9.70´10-19 M. Control experiments verified the specificity of the biosensor in the presence of mismatched DNA sequence. The DNA hybridization was monitored using a new ruthenium complex [Ru(dppz2(qtpyCl2; dppz = dipyrido [3,2–a:2’,3’-c] phenazine; qtpy=2,2’,-4,4”.4’4”’-quarterpyridyl redox indicator.

  16. Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Ensafi, Ali A., E-mail: ensafi@cc.iut.ac.ir [Department of Chemistry, Isfahan University of Technology, Isfahan (Iran, Islamic Republic of); Taei, M. [Department of Chemistry, Isfahan University of Technology, Isfahan (Iran, Islamic Republic of); Rahmani, H.R. [Department of Animal Science, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Khayamian, T. [Department of Chemistry, Isfahan University of Technology, Isfahan (Iran, Islamic Republic of)

    2011-10-01

    Highlights: > Chronic lymphocytic leukemia causes an increase in the number of white blood cells. > We introduced a highly sensitive biosensor for the detection of chronic lymphocytic leukemia. > A suitable 25-mer ssDNA probe was immobilized on the surface of the gold nanoparticles. > We used electrochemical impedance spectroscopy as a suitable tool for the detection. > Detection of chronic lymphocytic leukemia in blood sample was checked using the sensor. - Abstract: A simple and sensitive DNA impedance sensor was prepared for the detection of chronic lymphocytic leukemia. The DNA electrochemical biosensor is worked based on the electrochemical impedance spectroscopic (EIS) detection of the sequence-specific DNA related to chronic lymphocytic leukemia. The ssDNA probe was immobilized on the surface of the gold nanoparticles. Compared to the bare gold electrode, the gold nanoparticles-modified electrode could improve the density of the probe DNA attachment and hence the sensitivity of the DNA sensor greatly. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were performed in a solution containing 1.0 mmol L{sup -1} K{sub 3}[Fe(CN){sub 6}]/K{sub 4}[Fe(CN){sub 6}] and 50 mmol L{sup -1} phosphate buffer saline pH 6.87 plus 50 mmol L{sup -1} KCl. In the CV studied, the potential was cycled from 0.0 to +0.65 V with a scan rate of 50 mV s{sup -1}. Using EIS, the difference of the electron transfer resistance ({Delta}R{sub et}) was linear with the logarithm of the complementary oligonucleotides sequence concentrations in the range of 7.0 x 10{sup -12}-2.0 x 10{sup -7} mol L{sup -1}, with a detection limit of 1.0 x 10{sup -12} mol L{sup -1}. In addition, the DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles.

  17. Phase Transformations in Cast Duplex Stainless Steels

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon-Jun [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    Duplex stainless steels (DSS) constitute both ferrite and austenite as a matrix. Such a microstructure confers a high corrosion resistance with favorable mechanical properties. However, intermetallic phases such as σ and χ can also form during casting or high-temperature processing and can degrade the properties of the DSS. This research was initiated to develop time-temperature-transformation (TTT) and continuous-cooling-transformation (CCT) diagrams of two types of cast duplex stainless steels, CD3MN (Fe-22Cr-5Ni-Mo-N) and CD3MWCuN (Fe-25Cr-7Ni-Mo-W-Cu-N), in order to understand the time and temperature ranges for intermetallic phase formation. The alloys were heat treated isothermally or under controlled cooling conditions and then characterized using conventional metallographic methods that included tint etching, and also using electron microscopy (SEM, TEM) and wavelength dispersive spectroscopy (WDS). The kinetics of intermetallic-phase (σ + χ) formation were analyzed using the Johnson-Mehl-Avrami (MA) equation in the case of isothermal transformations and a modified form of this equation in the case of continuous cooling transformations. The rate of intermetallic-phase formation was found to be much faster in CD3MWCuN than CD3MN due mainly to differences in the major alloying contents such as Cr, Ni and Mo. To examine in more detail the effects of these elements of the phase stabilities; a series of eight steel castings was designed with the Cr, Ni and Mo contents systematically varied with respect to the nominal composition of CD3MN. The effects of varying the contents of alloying additions on the formation of intermetallic phases were also studied computationally using the commercial thermodynamic software package, Thermo-Calc. In general, σ was stabilized with increasing Cr addition and χ by increasing Mo addition. However, a delicate balance among Ni and other minor elements such as N and Si also exists. Phase equilibria in DSS can be affected by

  18. Phase transformations in cast duplex stainless steels

    Science.gov (United States)

    Kim, Yoon-Jun

    Duplex stainless steels (DSS) constitute both ferrite and austenite as a matrix. Such a microstructure confers a high corrosion resistance with favorable mechanical properties. However, intermetallic phases such as sigma (sigma) and chi (chi) can also form during casting or high-temperature processing and can degrade the properties of the DSS. This research was initiated to develop time-temperature-transformation (TTT) and continuous-cooling-transformation (CCT) diagrams of two types of cast duplex stainless steels, CD3MN (Fe-22Cr-5Ni-Mo-N) and CD3MWCuN (Fe-25Cr-7Ni-Mo-W-Cu-N), in order to understand the time and temperature ranges for intermetallic phase formation. The alloys were heat treated isothermally or under controlled cooling conditions and then characterized using conventional metallographic methods that included tint etching, and also using electron microscopy (SEM, TEM) and wavelength dispersive spectroscopy (WDS). The kinetics of intermetallic-phase (sigma + chi) formation were analyzed using the Johnson-Mehl-Avrami (JMA) equation in the case of isothermal transformations and a modified form of this equation in the case of continuous cooling transformations. The rate of intermetallic-phase formation was found to be much faster in CD3MWCuN than CD3MN due mainly to differences in the major alloying contents such as Cr, Ni and Mo. To examine in more detail the effects of these elements of the phase stabilities, a series of eight steel castings was designed with the Cr, Ni and Mo contents systematically varied with respect to the nominal composition of CD3MN. The effects of varying the contents of alloying additions on the formation of intermetallic phases were also studied computationally using the commercial thermodynamic software package, Thermo-Calc. In general, a was stabilized with increasing Cr addition and chi by increasing Mo addition. However, a delicate balance among Ni and other minor elements such as N and Si also exists. Phase equilibria in

  19. Anthraquinone as a Redox Label for DNA: Synthesis, Enzymatic Incorporation, and Electrochemistry of Anthraquinone-Modified Nucleosides, Nucleotides, and DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Pohl, Radek; Horáková Brázdilová, Petra; Vidláková, Pavlína; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 50 (2011), s. 14063-14073 ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : anthraquinone * DNA * electrochemistry * nucleosides * oligonucleotides Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  20. A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

    Science.gov (United States)

    Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

    2017-01-01

    This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

  1. Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

    International Nuclear Information System (INIS)

    Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

    1991-01-01

    Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

  2. Graphene oxide modified light addressable potentiometric sensor and its application for ssDNA monitoring.

    Science.gov (United States)

    Jia, Yunfang; Yin, Xue-Bo; Zhang, Jia; Zhou, Shuang; Song, Meng; Xing, Ke-Li

    2012-12-21

    A light addressable potentiometric sensor (LAPS) is a kind of silicon based semiconductor sensor, and surface modification is a fundamental problem for its application in biological fields. Graphene oxide (GO) based biochemically activated LAPS were proposed, called GO-LAPS. The GO-LAPS were applied to monitoring single strand DNA (ssDNA) probe immobilization and its hybridization with complementary ssDNA molecules of different chain lengths (30, 21 and 14 base pairs, respectively). It was discovered that the curves of LAPS' currents versus analyte concentrations for ssDNA probe binding and the target ssDNA hybridization were different. Explanations were proposed based on the semiconductor's surface-electric-field-effect and the electrical properties of ssDNA molecule. Moreover, comparisons between GO-LAPS and LAPS without GO modification were carried out. Enhanced response currents of GO-LAPS were reported experimentally and analyzed theoretically based on X-ray photoelectron spectroscopy (XPS) of GO-LAPS. The limitation of target ssDNA monitoring was 1 pM to 10 nM, which suggested that this LAPS based platform could be developed as a sensitive means for short chain ssDNA detection.

  3. Pnc1p-mediated nicotinamide clearance modifies the epigenetic properties of rDNA silencing in Saccharomyces cerevisiae.

    Science.gov (United States)

    McClure, Julie M; Gallo, Christopher M; Smith, Daniel L; Matecic, Mirela; Hontz, Robert D; Buck, Stephen W; Racette, Frances G; Smith, Jeffrey S

    2008-10-01

    The histone deacetylase activity of Sir2p is dependent on NAD(+) and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD(+) and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD(+) concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD(+) salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD(+) concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss-Handler NAD(+) salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.

  4. Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Macíčková-Cahová, Hana; Pivoňková, Hana; Špaček, Jan; Havran, Luděk; Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 9, č. 5 (2011), s. 1366-1371 ISSN 1477-0520 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk(CZ) LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : DNA tail-labelling * protein-DNA binding * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 3.696, year: 2011

  5. Voltammetric behavior of osmium-labeled DNA at mercury meniscus-modified solid amalgam electrodes. Detecting DNA hybridization

    Czech Academy of Sciences Publication Activity Database

    Josypčuk, Bohdan; Fojta, Miroslav; Havran, Luděk; Heyrovský, Michael; Paleček, Emil

    2006-01-01

    Roč. 18, č. 2 (2006), s. 186-194 ISSN 1040-0397 R&D Projects: GA MPO 1H-PK/42; GA AV ČR IAA4004402; GA AV ČR KJB4004302; GA AV ČR IBS5004355 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z50040507 Keywords : voltammetry * solid amalgam electrodes * DNA-osmium complex * hybridization * catalytic hydrogen evolution Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.444, year: 2006

  6. DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

    Directory of Open Access Journals (Sweden)

    Bodo C. Melnik

    2017-09-01

    Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

  7. Thermal stabilisation of RNA·RNA duplexes and G-quadruplexes by phosphorothiolate linkages.

    Science.gov (United States)

    Piperakis, Michael M; Gaynor, James W; Fisher, Julie; Cosstick, Richard

    2013-02-14

    The effect of 3'-S-phosphorothiolate linkages on the stability of RNA·RNA duplexes and G-quadruplex structures has been studied. 3'-Thio-2'-deoxyuridine was incorporated into RNA duplexes and thermal melting studies revealed that the resulting 3'-S-phosphorothiolate linkages increased the stability of the duplex to thermal denaturation. Additionally, and contrary to expectation, a similar effect on duplex stability was observed when the same thionucleoside was incorporated into the RNA strand of a RNA·DNA duplex. A suitably protected derivative of 3'-thio-2'-deoxyguanosine was prepared using an oxidation-reduction strategy and this residue also increased the thermal stability the [d(TGGGGT)](4) G-quadruplex when positioned centrally. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and imply that the previously noted high thermal stability of parallel RNA quadruplexes is not derived from H-bonding interactions of the 2'-hydroxyl group, but can be attributed to conformational effects.

  8. Practical, real-time, full duplex wireless

    KAUST Repository

    Jain, Mayank

    2011-01-01

    This paper presents a full duplex radio design using signal inversion and adaptive cancellation. Signal inversion uses a simple design based on a balanced/unbalanced (Balun) transformer. This new design, unlike prior work, supports wideband and high power systems. In theory, this new design has no limitation on bandwidth or power. In practice, we find that the signal inversion technique alone can cancel at least 45dB across a 40MHz bandwidth. Further, combining signal inversion cancellation with cancellation in the digital domain can reduce self-interference by up to 73dB for a 10MHz OFDM signal. This paper also presents a full duplex medium access control (MAC) design and evaluates it using a testbed of 5 prototype full duplex nodes. Full duplex reduces packet losses due to hidden terminals by up to 88%. Full duplex also mitigates unfair channel allocation in AP-based networks, increasing fairness from 0.85 to 0.98 while improving downlink throughput by 110% and uplink throughput by 15%. These experimental results show that a re- design of the wireless network stack to exploit full duplex capability can result in significant improvements in network performance. © 2011 ACM.

  9. Energy balance and deformation mechanisms of duplexes

    Science.gov (United States)

    Mitra, Gautam; Boyer, Steven E.

    A duplex consists of a series of imbricate faults that are asymptotic to a roof thrust and a floor thrust. Depending on the final orientations of the imbricate faults and the final position of the branch lines, a duplex may be hinterland-dipping, foreland-dipping, or an antiformal stack. The exact geometry depends on various factors such as the initial dimensions of the individual slices (horses), their lithology, the amount of displacement (normalized to size of horse) on each fault, and the mechanics of movement along each fault. The energy required in duplex formation can be determined by calculating the total work involved in emplacing each horse: this is given by where W t=W p+W b+W g+W iWp is the work involved in initiating and propagating a fracture. Wb is the work involved in basal sliding, which may be frictional or some form of ductile flow, Wg is the work done against gravity during the emplacement of the horse, and Wi is the work involved in the internal deformation of the horse. By calculating and comparing these work terms it is possible to predict the conditions under which the different types of duplexes will form. Normally, the development of a hinterland-dipping duplex is most likely. However, if deformation conditions are favorable, displacements on individual imbricate faults may be very large compared to the size of the horses, leading to the formation of either antiformal stacks or foreland-dipping duplexes.

  10. DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis.

    Science.gov (United States)

    Saeed, Ayman Ali; Sánchez, Josep Lluís Acero; O'Sullivan, Ciara K; Abbas, Mohammed Nooredeen

    2017-12-01

    Two different DNA (ERBB2c and CD24c) modified gold nanoparticles and graphene oxide loaded on glassy carbon electrodes were prepared for early detection of breast cancer markers by electrochemical detection of HER2. Comparative study of ERBB2c and CD24c for the detection was carried out. A "sandwich-type" detection strategy was employed in this electrochemical DNA biosensor and its response was measured by amperometric detection. The electrochemical signal enhancement achieved via gold nanoparticles and grapheme oxide system allowed for sensitive detection of the breast cancer biomarker ERBB2 and the control marker CD24. The modified graphene oxide was characterised using Raman spectroscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy transmission electron microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The various steps involved in the modification of a glassy carbon electrode with graphene oxide, gold nanoparticles and DNA probes, target and reporter probe were electrochemically characterised using cyclic voltammetry and electrochemical impedance spectroscopy. Using amperometric detection of a horse radish peroxidase label, detection limits of 0.16nM and 0.23nM were obtained with sensitivity 378nA/nM and 219nA/nM for ERBB2 andCD24 respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

    Science.gov (United States)

    Liao, Wei-Ching; Chuang, Min-Chieh; Ho, Ja-An Annie

    2013-12-15

    Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. DNA-repair measurements by use of the modified comet assay

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia

    2013-01-01

    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating...... laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell...

  13. Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

    International Nuclear Information System (INIS)

    Muller, E.

    2002-01-01

    Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

  14. Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs.

    Science.gov (United States)

    Benvidi, Ali; Tezerjani, Marzieh Dehghan; Jahanbani, Shahriar; Mazloum Ardakani, Mohammad; Moshtaghioun, Seyed Mohammad

    2016-01-15

    In this research, we have developed lable free DNA biosensors based on modified glassy carbon electrodes (GCE) with reduced graphene oxide (RGO) and carbon nanotubes (MWCNTs) for detection of DNA sequences. This paper compares the detection of BRCA1 5382insC mutation using independent glassy carbon electrodes (GCE) modified with RGO and MWCNTs. A probe (BRCA1 5382insC mutation detection (ssDNA)) was then immobilized on the modified electrodes for a specific time. The immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were performed under optimum conditions using different electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The proposed biosensors were used for determination of complementary DNA sequences. The non-modified DNA biosensor (1-pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS)/GCE), revealed a linear relationship between ∆Rct and logarithm of the complementary target DNA concentration ranging from 1.0×10(-16)molL(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.992, for DNA biosensors modified with multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (RGO) wider linear range and lower detection limit were obtained. For ssDNA/PANHS/MWCNTs/GCE a linear range 1.0×10(-17)mol L(-1)-1.0×10(-10)mol L(-1) with a correlation coefficient of 0.993 and for ssDNA/PANHS/RGO/GCE a linear range from 1.0×10(-18)mol L(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.985 were obtained. In addition, the mentioned biosensors were satisfactorily applied for discriminating of complementary sequences from noncomplementary sequences, so the mentioned biosensors can be used for the detection of BRCA1-associated breast cancer. Copyright © 2015. Published by Elsevier B.V.

  15. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Science.gov (United States)

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  16. Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

    Science.gov (United States)

    Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

    2006-08-14

    Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

  17. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2006-08-01

    Full Text Available Abstract Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was

  18. Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer systems for applications in genetically modified organisms analysis.

    Science.gov (United States)

    Gonzalez García, Eric; Ressmann, Anna K; Gaertner, Peter; Zirbs, Ronald; Mach, Robert L; Krska, Rudolf; Bica, Katharina; Brunner, Kurt

    2014-12-01

    To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method.

  19. Fabrication of chronocoulometric DNA sensor based on gold nanoparticles/poly(l-lysine) modified glassy carbon electrode.

    Science.gov (United States)

    Wang, Jie; Zhang, Shaojun; Zhang, Yuzhong

    2010-01-15

    In this work, we fabricated a sensitivity chronocoulometric DNA sensor (CDS) based on gold nanoparticles (AuNPs)/poly(L-lysine) complex film modified glassy carbon electrode. Hexaammineruthenium(III) chloride ([Ru(NH3)6]3+) was used as the electroactive indicator. The assembled process was investigated by cyclic voltammetry (CV) and chronocoulometry (CC). CC is used to monitor the DNA hybridization event by measurement of electrostatic binding [Ru(NH3)6]3+. Under the optimal conditions, the signal of [Ru(NH3)6]3+ was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 x 10(-13) to 1.0 x 10(-11) M, and the detection limit is 3.5 x 10(-14) M.

  20. Law of mixture used to model the flow behavior of a duplex stainless steel at high temperatures

    International Nuclear Information System (INIS)

    Momeni, A.; Dehghani, K.; Poletti, M.C.

    2013-01-01

    In this investigation the flow curves of a duplex stainless steel were drawn by performing hot compression tests over a wide temperature range of 950–1200 °C and strain rates of 0.001–100 s −1 . The flow curves of ferrite and austenite phases in the duplex structure were depicted by conducting similar hot compression tests on two steels that were cast and prepared with the same chemical compositions. The flow curves of the austenitic steel were found typical of dynamic recrystallization. They were successfully modeled by using the experimental exponential equation proposed by Cingara and McQueen. The flow curves of the ferritic steel were typical of dynamic recovery. They were modeled by the dislocation density evolution function proposed by Estrin and Meckning. Comparing the flow curves of three studied steels, it was found that the flow curves of the duplex steel were very similar and close to those of the ferrite steel. It was understood that in a duplex structure of ferrite and austenite the flow behavior is mostly controlled by the softer phase, i.e. ferrite. The law of mixture was modified to consider the strain partitioning between ferrite and austenite. The distribution coefficients of ferrite and austenite were described and determined at different deformation conditions. The results of modeling satisfactorily predicted the experimental curves. It was shown that the influence of austenite on the flow behavior of the duplex structure is almost low. However, it increases as strain rate or temperature rises. - Highlights: ► Flow curves of austenite and ferrite in the duplex steel were modeled separately. ► The flow behavior of the duplex steel is mostly controlled by ferrite. ► The effect of austenite on flow curve increases with temperature and strain rate. ► The flow curve of the duplex steel is modeled by the modified law of mixture

  1. DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

    Science.gov (United States)

    Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

    2015-05-01

    Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A modified method for whole exome resequencing from minimal amounts of starting DNA.

    Directory of Open Access Journals (Sweden)

    Iwanka Kozarewa

    Full Text Available Next generation DNA sequencing (NGS technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap method generate datasets that are comparable to standard amount (SA whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.

  3. A modified method for whole exome resequencing from minimal amounts of starting DNA.

    Science.gov (United States)

    Kozarewa, Iwanka; Rosa-Rosa, Juan Manuel; Wardell, Christopher P; Walker, Brian A; Fenwick, Kerry; Assiotis, Ioannis; Mitsopoulos, Costas; Zvelebil, Marketa; Morgan, Gareth J; Ashworth, Alan; Lord, Christopher J; J, Christopher

    2012-01-01

    Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.

  4. Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

    OpenAIRE

    Parsons, C A; West, S C

    1990-01-01

    T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

  5. 2-Aminopurine-modified DNA homopolymers for robust and sensitive detection of mercury and silver.

    Science.gov (United States)

    Zhou, Wenhu; Ding, Jinsong; Liu, Juewen

    2017-01-15

    Heavy metal detection is a key topic in analytical chemistry. DNA-based metal recognition has advanced significantly producing many specific metal ligands, such as thymine for Hg 2+ and cytosine for Ag + . For practical applications, however, robust sensors that can work in a diverse range of salt concentrations need to be developed, while most current sensing strategies cannot meet this requirement. In this work, 2-aminopurine (2AP) is used as a fluorescence label embedded in the middle of four 10-mer DNA homopolymers. 2AP can be quenched up to 98% in these DNA without an external quencher. The interaction between 2AP and all common metal ions is studied systematically for both free 2AP base and 2AP embedded DNA homopolymers. With such low background, Hg 2+ induces up to 14-fold signal enhancement for the poly-T DNA, and Ag + enhances up to 10-fold for the poly-C DNA. A detection limit of 3nM is achieved for both metals. With these four probes, silver and mercury can be readily discriminated from the rest. A comparison with other signaling methods was made using fluorescence resonance energy transfer, graphene oxide, and SYBR Green I staining, respectively, confirming the robustness of the 2AP label. Detection of Hg 2+ in Lake Huron water was also achieved with a similar sensitivity. This work has provided a comprehensive fundamental understanding of using 2AP as a label for metal detection, and has achieved the highest fluorescence enhancement for non-protein targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP by Assimilating Probes

    Directory of Open Access Journals (Sweden)

    Ryo Kubota

    2015-03-01

    Full Text Available Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.

  7. Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Ma, Xingyi; Cao, Cuong

    2011-01-01

    In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target...... DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency...... in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which...

  8. Synthesis of Hydrazone-Modified Nucleotides and Their Polymerase Incorporation onto DNA for Redox Labeling

    Czech Academy of Sciences Publication Activity Database

    Raindlová, Veronika; Pohl, Radek; Klepetářová, Blanka; Havran, Luděk; Šimková, Eva; Horáková Brázdilová, Petra; Pivoňková, Hana; Fojta, Miroslav; Hocek, Michal

    2012-01-01

    Roč. 77, č. 8 (2012), s. 652-662 ISSN 2192-6506 R&D Projects: GA AV ČR(CZ) IAA400040901; GA ČR GBP206/12/G151 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040702 Keywords : DNA * oligonucleotides * polymerase * hydrazones * nucleotides Subject RIV: CC - Organic Chemistry

  9. Detection of genetically modified organisms (GMOs using isothermal amplification of target DNA sequences

    Directory of Open Access Journals (Sweden)

    La Mura Maurizio

    2009-02-01

    Full Text Available Abstract Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR. Here we have applied the loop-mediated isothermal amplification (LAMP method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  10. DNA Detection by Flow Cytometry using PNA‐Modified Metal–Organic Framework Particles

    NARCIS (Netherlands)

    Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon; Corradini, Roberto; Huskens, J.

    2017-01-01

    A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal–organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or

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

    Directory of Open Access Journals (Sweden)

    Ceren Sengiz

    2015-09-01

    Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

  12. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

    Directory of Open Access Journals (Sweden)

    Vagner Laura L

    2008-05-01

    Full Text Available Abstract Background Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking. Results Using degenerative primers and the rapid amplification of cDNA ends (RACE procedure, we cloned the Duplex-Specific Nuclease (DSN gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5 and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact. Conclusion We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate

  13. Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae

    Directory of Open Access Journals (Sweden)

    Hilary A. McManus

    2017-05-01

    Full Text Available Background Comparative studies of chloroplast genomes (plastomes across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae, one from Hydrodictyon reticulatum and the other from Pediastrum duplex. Methods Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed. Results The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae. Discussion The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique

  14. Full Duplex Communications in 5G Small Cells

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Gatnau, Marta; Berardinelli, Gilberto

    2017-01-01

    Full duplex communication promises system performance improvement over conventional half duplex communication by allowing simultaneous transmission and reception. However, such concurrent communication results in strong self interference and an increase in the overall network interference, and ca...

  15. Preparation of carboxyl group-modified palladium nanoparticles in an aqueous solution and their conjugation with DNA

    Science.gov (United States)

    Wang, Zhifei; Li, Hongying; Zhen, Shuang; He, Nongyue

    2012-05-01

    The use of nanomaterials in biomolecular labeling and their corresponding detection has been attracting much attention, recently. There are currently very few studies on palladium nanoparticles (Pd NPs) due to their lack of appropriate surface functionalities for conjugation with DNA. In this paper, we thus firstly present an approach to prepare carboxyl group-modified Pd NPs (with an average size of 6 nm) by the use of 11-mercaptoundecanoic acid (MUDA) as a stabilizer in the aqueous solution. The effect of the various reducing reaction conditions on the morphology of the Pd NPs was investigated. The particles were further characterized by TEM, UV-vis, FT-IR and XPS techniques. DNA was finally covalently conjugated to the surface of the Pd NPs through the activation of the carboxyl group, which was confirmed by agarose gel electrophoresis and fluorescence analysis. The resulting Pd NPs-DNA conjugates show high single base pair mismatch discrimination capabilities. This work therefore sets a good foundation for further applications of Pd NPs in bio-analytical research.

  16. Fluoroquinolone-Gyrase-DNA Complexes

    Science.gov (United States)

    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

    2014-01-01

    DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 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-Gly81 and GyrB-Glu466 residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases. PMID:24497635

  17. Identification of Clinical Isolates of Candida using Duplex PCR

    Directory of Open Access Journals (Sweden)

    Homeyra Babaei

    2016-11-01

    Full Text Available Abstract Background: Candida albicans is still the main etiologic agent of candidiasis. However, infections of non-albicans Candida species are increasing. Candida dubliniensis is similar to C. albicans phenotypically and must be identified due to the better management of infection. The aim of the present study is to defferentiate and identify Candida species by Duplex PCR for get-ting an epidemiological data of Candida species among clinical specimens. Materials and Methods: DNA was extracted using phenol-chloroform method from fresh colonies. Internal Transcribed Spacer region was amplified by polymerase chain reaction using specific primers. Based on differences of bands sizes on agarose gel electrophoresis, species were identified. Results: Ninety four out of 100 patients (49 males and 51 females had predisposing fac-tors in the present study. Diabetes (73.4%, use of antibiotic (6.3%, vitamin deficiency (4.3% were the main predisposing factors. The most specimens belonged to mouth (75%, vagina (5%, and blood (4%. All isolates were identified as C. albicans. Conclusion: Duplex PCR is a rapid and precise method for the detection and differentia-tion of Candida species carefully, and in this method, phenotypic tests like germ-tube and chla-mydoconidia production, as well as biochemical tests are not required for clinical laboratories that have limited resources and time for response to the patients, and it can replace with the traditional methods.

  18. Simultaneous Extraction of DNA and RNA from Animal Cells by a Modified Laemmli Buffer

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Nasrin Shojaie, Mahmood S. Ghaffari & Zahra Safari ### Abstract Simultaneous investigation of DNA and RNA is often a necessity in genetic manipulation and biological researches. Besides, most of the traditional procedures devised for RNA isolation have some difficulties associated with RNase activity. Therefore, this protocol presents a safer process to extract high purity RNA in shorter time. ### Introduction The protocol describes a simple and less time-consuming m...

  19. DNA interactions of 2-pyrrolinodoxorubicin, a distinctively more potent daunosamine-modified analogue of doxorubicin

    Czech Academy of Sciences Publication Activity Database

    Štěpánková, Jana; Studenovský, Martin; Malina, Jaroslav; Kašpárková, J.; Lišková, Barbora; Nováková, Olga; Ulbrich, Karel; Brabec, Viktor

    2011-01-01

    Roč. 82, č. 3 (2011), s. 227-235 ISSN 0006-2952 R&D Projects: GA ČR(CZ) GD301/09/H004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40500505 Keywords : doxorubicin * 2-pyrrolinodoxorubicin * DNA Subject RIV: BO - Biophysics Impact factor: 4.705, year: 2011

  20. Application of chemically modified oligonucleotides in nanopore sensing and DNA nano – biotechnology

    OpenAIRE

    Mitchell, N. J.

    2010-01-01

    This thesis describes how targeted chemical modification can enhance the properties of nucleic acids for use in (i) nanopore analytics and (ii) nanobiotechnology. In nanopore analytics, individual molecules are detected as they pass a nanoscale pore to give rise to detectable blockades in ionic current. Despite progress in the sensing of a multitude of molecular species, the analytical resolution in the sensing of DNA is poor as individual bases in passing strands cannot be res...

  1. Electrochemical biosensor modified with dsDNA monolayer for restriction enzyme activity determination.

    Science.gov (United States)

    Zajda, Joanna; Górski, Łukasz; Malinowska, Elżbieta

    2016-06-01

    A simple and cost effective method for the determination of restriction endonuclease activity is presented. dsDNA immobilized at a gold electrode surface is used as the enzymatic substrate, and an external cationic redox probe is employed in voltammetric measurements for analytical signal generation. The assessment of enzyme activity is based on a decrease of a current signal derived from reduction of methylene blue which is present in the sample solution. For this reason, the covalent attachment of the label molecule is not required which significantly reduces costs of the analysis and simplifies the entire determination procedure. The influence of buffer components on utilized dsDNA/MCH monolayer stability and integrity is also verified. Electrochemical impedance spectroscopy measurements reveal that due to pinhole formation during enzyme activity measurement the presence of any surfactants should be avoided. Additionally, it is shown that the sensitivity of the electrochemical biosensor can be tuned by changing the restriction site location along the DNA length. Under optimal conditions the proposed biosensor exhibits a linear response toward PvuII activity within a range from 0.25 to 1.50 U/μL. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Simultaneous DPV determination of morphine and codeine using dsDNA modified screen printed electrode strips coupled with electromembrane extraction

    Directory of Open Access Journals (Sweden)

    Rouhollah Feizbakhsh

    2016-01-01

    Full Text Available In this work a sensitive electrochemical sensor for simultaneous determination of morphine and codeine constructed by application of disposable screen printed carbon electrode strips (SPCE modified by double strand (ds calf thymus DNA. According to the results of the modified SPCE strips and experimented parameters, we observed a considerable shift between potentials of morphine and codeine current peaks. Related to these observed shifts, we studied on the effect of the concentration of modifier and pH value on the anodic oxidation pattern of morphine and codeine in the case of optimize the method to get better signals with maximum potential distance. Also to boosting the LODs of this electroanalytical method coupled with an electro-membrane preconcentration (EME step. The calibration curve which was plotted by the variation of differential pulse voltammetry (DPV currents as a function of different morphine and codeine concentration were linear within the range of 0.7– 40 µM and 2.3- 40 µM for morphine and codeine respectively. Also the limits of detection were 0.07 µM and 0.23 µM, respectively. Finally, the proposed method was able to determine morphine and codeine simultaneously and effectively in urinary real samples

  3. Duplex scanning using sparse data sequences

    DEFF Research Database (Denmark)

    Møllenbach, S. K.; Jensen, Jørgen Arendt

    2008-01-01

    The velocity distribution in vessels can be displayed using duplex scanning where B-mode acquisitions are interspaced with the velocity data. This gives an image for orientation, but lowers the maximum detectable velocity by a factor of two. Other pulse sequences either omits the B-mode image...... is scaled by the factor A/T. The approach has been investigated using in vivo RF data from the Hepatic vein, Carotid artery and Aorta from a 33 year old healthy male. A B-K Medical 3535 ultrasound scanner has been used in Duplex mode with a BK 8556, 3.2 MHz linear array probe. The sampling frequency...

  4. Full-duplex optical communication system

    Science.gov (United States)

    Shay, Thomas M. (Inventor); Hazzard, David A. (Inventor); Horan, Stephen (Inventor); Payne, Jason A. (Inventor)

    2004-01-01

    A method of full-duplex electromagnetic communication wherein a pair of data modulation formats are selected for the forward and return data links respectively such that the forward data electro-magnetic beam serves as a carrier for the return data. A method of encoding optical information is used wherein right-hand and left-hand circular polarizations are assigned to optical information to represent binary states. An application for an earth to low earth orbit optical communications system is presented which implements the full-duplex communication and circular polarization keying modulation format.

  5. Color coded duplex sonography. Interdisciplinary vascular ultrasonography

    International Nuclear Information System (INIS)

    Kubale, R.; Stiegler, H.

    2002-01-01

    An interdisciplinary team of experts impart the state of the art in color coded duplex sonography applied for examination of all anatomic areas. Detailed information is given for every vascular area, describing the examination procedure, possible origins of mistakes or faults, and means to avoid them. In addition to the classical applications, eg. for examination of the extra- and intracranial vessels, vessels of the limbs, visceral vessels and the vessels of liver and kidney, there are chapters devoted to: haemodialysis shunts, arteriovenous malformations, duplex sonography of the penis, tumor vascularisation. (orig./CB) [de

  6. Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

    Directory of Open Access Journals (Sweden)

    Satoshi Obika

    2010-11-01

    Full Text Available Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

  7. Theoretical modelling of epigenetically modified DNA sequences [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Alexandra Teresa Pires Carvalho

    2015-05-01

    Full Text Available We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM and hybrid quantum mechanics/molecular mechanics (QM/MM methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.

  8. Recognition of DNA modified with antitumor cisplatin by p53-family proteins

    Czech Academy of Sciences Publication Activity Database

    Pivoňková, Hana; Fojta, Miroslav

    2009-01-01

    Roč. 276, č. 1 (2009), s. 265-266 ISSN 1742-464X. [34th FEBS Congress. 04.07.2009-09.07.2009, Prague] R&D Projects: GA AV ČR(CZ) IAA500040701; GA ČR(CZ) GP204/07/P476; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : p53-family proteins * interaction * DNA modification Subject RIV: BO - Biophysics

  9. Voltammetric behavior of DNA modified with osmium tetroxide 2,2'-bipyridine at mercury electrodes

    Czech Academy of Sciences Publication Activity Database

    Havran, Luděk; Fojta, Miroslav; Paleček, Emil

    2004-01-01

    Roč. 63, 1-2 (2004), s. 239-243 ISSN 1567-5394 R&D Projects: GA AV ČR IAA4004108; GA AV ČR KJB4004302; GA ČR GA204/00/D049; GA ČR GA204/03/0566; GA AV ČR IBS5004107; GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z5004920 Keywords : chemical modification of DNA * osmium tetroxide complexes * cyclic voltammetry Subject RIV: BO - Biophysics Impact factor: 2.261, year: 2004

  10. Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

    Science.gov (United States)

    Ordonez, Heather; Unciuleac, Mihaela; Shuman, Stewart

    2012-05-01

    The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

  11. The Modular Construction of DNA Double Helix

    Indian Academy of Sciences (India)

    DNA or the left-handed double helix,. Z- DNA. Construction of the Module .... 12. DNA, namely, replication and transcription. In the former case, Figure 3. 8 would represent a DNA single strand-generated by splitting of the mother duplex ...

  12. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food.

    Science.gov (United States)

    Jiang, Xiaohua; Zhang, Huimin; Wu, Jun; Yang, Xiang; Shao, Jingwei; Lu, Yujing; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2014-10-01

    In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS(2-) as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS(2-) with H2O2. Then the linear range is from 0.05 to 0.5 μM while detection limit is 5nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. DNA microarrays on ultraviolet-modified surfaces for speciation of bacteria.

    Science.gov (United States)

    Liu, Yanyang; He, Jianzhong; Yang, Kun-Lin

    2014-02-15

    In this study, we report an approach to activate inert hydrocarbon monolayers with ultraviolet (UV) light to fabricate DNA microarrays. Unlike traditional microarrays that require reactive functional groups on the surface, our DNA microarray is built on an inert layer of N,N-dimethyl-N-octadecyl(3-aminopropyl)trimethoxysilyl chloride silane (DMOAP). This layer is activated by UV (254 nm) just prior to the immobilization of oligonucleotide probes. Our X-ray photoelectron spectroscopy (XPS) results show that new functional groups such as alcohol (C--O), aldehyde (C=O), and carboxylic acid (O--C=O) form on the surface after the UV exposure. Among them, aldehyde groups are responsible for the immobilization of amine-label oligonucleotides. By using this approach, we further optimize UV exposure time and oligonucleotide concentration and also reduce agent concentration to achieve a high density of immobilized oligonucleotides up to 0.16 pmol/mm². As a proof of concept, we demonstrate that this microarray can be used for differentiation of different Clostridium species such as Clostridium acetobutylicum, Clostridium butylicum, and Clostridium beijerinkii. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. [Detection of recombinant-DNA in foods from stacked genetically modified plants].

    Science.gov (United States)

    Sorokina, E Iu; Chernyshova, O N

    2012-01-01

    A quantitative real-time multiplex polymerase chain reaction method was applied to the detection and quantification of MON863 and MON810 in stacked genetically modified maize MON 810xMON 863. The limit of detection was approximately 0,1%. The accuracy of the quantification, measured as bias from the accepted value and the relative repeatability standard deviation, which measures the intra-laboratory variability, were within 25% at each GM-level. A method verification has demonstrated that the MON 863 and the MON810 methods can be equally applied in quantification of the respective events in stacked MON810xMON 863.

  15. Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Xiaoming; Gelinas, Amy D.; von Carlowitz, Ira; Janjic, Nebojsa; Pyle, Anna Marie (Yale); (SomaLogic)

    2017-10-09

    IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.

  16. Au nanoparticle-modified DNA sensor based on simultaneous electrochemical impedance spectroscopy and localized surface plasmon resonance.

    Science.gov (United States)

    Cheng, Xin R; Hau, Ben Y H; Endo, Tatsuro; Kerman, Kagan

    2014-03-15

    Electrochemical impedance spectroscopy (EIS) and localized surface plasmon resonance (LSPR) were performed on the same Au nanoparticle (AuNP)-modified indium tin oxide (ITO) coated glass surfaces. Cyclic voltammetry was applied to electrodeposit AuNPs on ITO surface directly. The surface plasmon band characterization of AuNPs was initially studied by controlling the electrodeposition conditions. It was found that the size of AuNP clusters was significantly affected by the applied potential and KCl concentration in solution. The dual-detection platform was applied to detect DNA hybridization related to a specific point mutation in apolipoprotein E gene (ApoE), which was related to the progression of Alzheimer's disease. The preliminary results facilitate the development of a versatile biosensor that can be easily miniaturized and integrated into a high-throughput diagnostic device. © 2013 Elsevier B.V. All rights reserved.

  17. Ultra-short silicon MMI duplexer

    Science.gov (United States)

    Yi, Huaxiang; Huang, Yawen; Wang, Xingjun; Zhou, Zhiping

    2012-11-01

    The fiber-to-the-home (FTTH) systems are growing fast these days, where two different wavelengths are used for upstream and downstream traffic, typically 1310nm and 1490nm. The duplexers are the key elements to separate these wavelengths into different path in central offices (CO) and optical network unit (ONU) in passive optical network (PON). Multimode interference (MMI) has some benefits to be a duplexer including large fabrication tolerance, low-temperature dependence, and low-polarization dependence, but its size is too large to integrate in conventional case. Based on the silicon photonics platform, ultra-short silicon MMI duplexer was demonstrated to separate the 1310nm and 1490nm lights. By studying the theory of self-image phenomena in MMI, the first order images are adopted in order to keep the device short. A cascaded MMI structure was investigated to implement the wavelength splitting, where both the light of 1310nm and 1490nm was input from the same port, and the 1490nm light was coupling cross the first MMI and output at the cross-port in the device while the 1310nm light was coupling through the first and second MMI and output at the bar-port in the device. The experiment was carried on with the SOI wafer of 340nm top silicon. The cascaded MMI was investigated to fold the length of the duplexer as short as 117μm with the extinct ratio over 10dB.

  18. Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin

    Czech Academy of Sciences Publication Activity Database

    Vrána, Oldřich; Mašek, Vlastimil; Dražan, Viktor; Brabec, Viktor

    2007-01-01

    Roč. 159, č. 1 (2007), s. 1-8 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA203/05/2032; GA ČR(CZ) GD204/03/H016; GA ČR(CZ) GA305/05/2030; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA MŠk(CZ) LC06030; GA MŠk(CZ) 1P05OC070; GA MŠk(CZ) 1P05OC072 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * platinum * cancer Subject RIV: BO - Biophysics Impact factor: 3.677, year: 2007

  19. Relaxed specificity of prokaryotic DNA methyltransferases results in DNA site-specific modification of RNA/DNA heteroduplexes.

    Science.gov (United States)

    Wons, Ewa; Mruk, Iwona; Kaczorowski, Tadeusz

    2015-11-01

    RNA/DNA hybrid duplexes regularly occur in nature, for example in transcriptional R loops. Their susceptibility to modification by DNA-specific or RNA-specific enzymes is, thus, a biologically relevant question, which, in addition, has possible biotechnological implications. In this study, we investigated the activity of four isospecific DNA methyltransferases (M.EcoVIII, M.LlaCI, M.HindIII, M.BstZ1II) toward an RNA/DNA duplex carrying one 5'-AAGCUU-3'/3'-TTCGAA-5' target sequence. The analyzed enzymes belong to the β-group of adenine N6-methyltransferases and recognize the palindromic DNA sequence 5'-AAGCTT-3'/3'-TTCGAA-5'. Under standard conditions, none of these isospecific enzymes could detectibly methylate the RNA/DNA duplex. However, the addition of agents that generally relax specificity, such as dimethyl sulfoxide (DMSO) and glycerol, resulted in substantial methylation of the RNA/DNA duplex by M.EcoVIII and M.LlaCI. Only the DNA strand of the RNA/DNA duplex was methylated. The same was not observed for M.HindIII or M.BstZ1II. This is, to our knowledge, the first report that demonstrates such activity by prokaryotic DNA methyltransferases. Possible applications of these findings in a laboratory practice are also discussed.

  20. Combinatorial treatment of DNA and chromatin-modifying drugs cause cell death in human and canine osteosarcoma cell lines.

    Directory of Open Access Journals (Sweden)

    Venugopal Thayanithy

    Full Text Available Downregulation of microRNAs (miRNAs at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat and the DNA methylation inhibitor Zebularine (Zeb, with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

  1. Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

    Energy Technology Data Exchange (ETDEWEB)

    Lucarelli, Fausto; Marrazza, Giovanna; Palchetti, Ilaria; Cesaretti, S.; Mascini, Marco

    2002-09-26

    This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence. The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised. The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.

  2. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  3. Knots modify the coil-stretch transition in linear DNA polymers.

    Science.gov (United States)

    Soh, Beatrice W; Narsimhan, Vivek; Klotz, Alexander R; Doyle, Patrick S

    2018-02-28

    We perform single-molecule DNA experiments to investigate the relaxation dynamics of knotted polymers and examine the steady-state behavior of knotted polymers in elongational fields. The occurrence of a knot reduces the relaxation time of a molecule and leads to a shift in the molecule's coil-stretch transition to larger strain rates. We measure chain extension and extension fluctuations as a function of strain rate for unknotted and knotted molecules. The curves for knotted molecules can be collapsed onto the unknotted curves by defining an effective Weissenberg number based on the measured knotted relaxation time in the low extension regime, or a relaxation time based on Rouse/Zimm scaling theories in the high extension regime. Because a knot reduces a molecule's relaxation time, we observe that knot untying near the coil-stretch transition can result in dramatic changes in the molecule's conformation. For example, a knotted molecule at a given strain rate can experience a stretch-coil transition, followed by a coil-stretch transition, after the knot partially or fully unties.

  4. HIV TAT peptide modifies the distribution of DNA nanolipoparticles following convection-enhanced delivery.

    Science.gov (United States)

    MacKay, J Andrew; Li, Weijun; Huang, Zhaohua; Dy, Edward E; Huynh, Grace; Tihan, Tarik; Collins, Rodney; Deen, Dennis F; Szoka, Francis C

    2008-05-01

    We evaluated gene transfer using PEGylated bioresponsive nanolipid particles (NLPs) containing plasmid DNA administered by convection-enhanced delivery (CED) into orthotopically implanted U87-MG tumors in rat brain. We hypothesized that attachment of the human immunodeficiency virus trans-acting transcriptional activator peptide (TATp) to pH-sensitive, reduction-sensitive NLPs would increase gene transfer. TATp was attached either directly to a phospholipid (TATp-lipid) or via a 2-kd polyethylene glycol (PEG) to a lipid (TATp-PEG-lipid). Incorporation of 0.3 mol% TATp-PEG into pH-sensitive NLPs improved transfection 100,000-fold compared to NLPs in culture. In the brain or implanted tumors, the TATp-PEG restricted NLP convection to regions adjacent to the infusion catheter. Gene transfer in the brain from TATp-PEG NLPs, measured by green fluorescent protein (GFP) expression, was substantially greater than from NLPs adjacent to the catheter. Gene transfer using TATp-PEG NLPs, measured by luciferase expression, was 8-12-fold greater than from a 1,2-dioleoyl-3-trimethylammonium-propane/cholesterol cationic lipoplex but 13-27-fold less than from the NLPs. Brain luciferase expression was localized in perivascular macrophages. Thus a cationic ligand, such as the TATp-PEG-lipid, can dramatically increase gene expression in culture, in the normal brain, and in implanted tumors; however, restriction of NLP distribution to the vicinity of the infusion catheter reduces the absolute level of gene transfer.

  5. Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

    Science.gov (United States)

    Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

    2014-07-05

    The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the

  6. Detection of genetically modified DNA in fresh and processed foods sold in Kuwait.

    Science.gov (United States)

    Al-Salameen, Fadila; Kumar, Vinod; Al-Aqeel, Hamed; Al-Hashash, Hanadi; Hejji, Ahmed Bin

    2012-01-01

    Developments in genetic engineering technology have led to an increase in number of food products that contain genetically engineered crops in the global market. However, due to lack of scientific studies, the presence of genetically modified organisms (GMOs) in the Kuwaiti food market is currently ambiguous. Foods both for human and animal consumption are being imported from countries that are known to produce GM food. Therefore, an attempt has been made to screen foods sold in the Kuwaiti market to detect GMOs in the food. For this purpose, samples collected from various markets in Kuwait have been screened by SYBR green-based real time polymerase chain reaction (RT-PCR) method. Further confirmation and GMO quantification was performed by TaqMan-based RT-PCR. Results indicated that a significant number of food commodities sold in Kuwait were tested positive for the presence of GMO. Interestingly, certain processed foods were tested positive for more than one transgenic events showing complex nature of GMOs in food samples. Results of this study clearly indicate the need for well-defined legislations and regulations on the marketing of approved GM food and its labeling to protect consumer's rights.

  7. Multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole DNA biosensor for label-free detection of genetically modified organisms by QCM and EIS.

    Science.gov (United States)

    Truong, Thi Ngoc Lien; Tran, Dai Lam; Vu, Thi Hong An; Tran, Vinh Hoang; Duong, Tuan Quang; Dinh, Quang Khieu; Tsukahara, Toshifumi; Lee, Young Hoon; Kim, Jong Seung

    2010-01-15

    In this paper, we describe DNA electrochemical detection for genetically modified organism (GMO) based on multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole (PPy). DNA hybridization is studied by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). An increase in DNA complementary target concentration results in a decrease in the faradic charge transfer resistance (R(ct)) and signifying "signal-on" behavior of MWCNTs-PPy-DNA system. QCM and EIS data indicated that the electroanalytical MWCNTs-PPy films were highly sensitive (as low as 4pM of target can be detected with QCM technique). In principle, this system can be suitable not only for DNA but also for protein biosensor construction.

  8. DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

    Science.gov (United States)

    Nzeduru, Chinyere V; Ronca, Sandra; Wilkinson, Mike J

    2012-01-01

    Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

  9. DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

    Directory of Open Access Journals (Sweden)

    Chinyere V Nzeduru

    Full Text Available Transgenes encoding for insecticidal crystal (Cry proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs represents a major element of the Environmental Risk Assessments (ERAs used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins. Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

  10. Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

    DEFF Research Database (Denmark)

    Yi, Sun; Perch-Nielsen, Ivan R.; Dufva, Martin

    2012-01-01

    -modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid...

  11. Comparative analysis of the end-joining activity of several DNA ligases.

    Directory of Open Access Journals (Sweden)

    Robert J Bauer

    Full Text Available DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1 DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs. This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.

  12. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    Science.gov (United States)

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of syndrome virus (WSSV) DNA was successfully demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Dúplex telefónico - Duplex telephonic

    Directory of Open Access Journals (Sweden)

    Santos López, Pascual

    2006-12-01

    Full Text Available Only 19 years after a timid introduction of telephony in Spain and seven years before the founding of the National Telephonic Company of Spain, Pascual Salmerón Gómez records his two patents: "Duplex telephonic" and "Simultaneous duplex telephonic with telegraph machines". Two high technical complexity patents, registered in 1917 in the town of Arucas, province of Gran Canaria analyzed to our readers.Nephew of the inventor of the "Float Salmerón," José Salmerón Rojas and brother of Gerónimo Gómez Salmerón, inventor of "Cerealémetro" and owner of the theater Borras and with which we ended the saga Salmerón.

  14. Diffraction study on aging duplex stainless steel

    International Nuclear Information System (INIS)

    Ishigaki, Toru; Miura, Takayuki; Kuwano, Hisashi; Torii, Syuuki; Kamiyama, Takashi

    2001-01-01

    SUS329J2L duplex stainless steels exhibit high strength and resistance to corrosion, so widely used in piping of industrial plants. However, it is known that they are brought deterioration of strength using for long time. This reason of this deterioration is that ferrite decomposes to Fe-rich α phase and Cr-rich α' phase and, Cr-rich α' phase decreases mechanical properties and resistance for corrosion. In this experiment, we made neutron diffraction experiments on long time aging (625 K, 16000 h) duplex stainless steel to observe the behavior for α phase and α' phase, using Sirius diffractometer at KENS. The result shows, the lattice parameters in α phase were decreased. In contrast to, its in austenite (γ phase) were slightly increased. (author)

  15. Duplex Tear Film Evaporation Analysis.

    Science.gov (United States)

    Stapf, M R; Braun, R J; King-Smith, P E

    2017-12-01

    Tear film thinning, hyperosmolarity, and breakup can cause irritation and damage to the human eye, and these form an area of active investigation for dry eye syndrome research. Recent research demonstrates that deficiencies in the lipid layer may cause locally increased evaporation, inducing conditions for breakup. In this paper, we explore the conditions for tear film breakup by considering a model for tear film dynamics with two mobile fluid layers, the aqueous and lipid layers. In addition, we include the effects of osmosis, evaporation as modified by the lipid, and the polar portion of the lipid layer. We solve the system numerically for reasonable parameter values and initial conditions and analyze how shifts in these cause changes to the system's dynamics.

  16. DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

    Science.gov (United States)

    Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

    2017-05-01

    Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

  17. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    Science.gov (United States)

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Joint duplex mode selection, channel allocation, and power control for full-duplex cognitive femtocell networks

    Directory of Open Access Journals (Sweden)

    Mingjie Feng

    2015-02-01

    Full Text Available In this paper, we aim to maximize the sum rate of a full-duplex cognitive femtocell network (FDCFN as well as guaranteeing the quality of service (QoS of users in the form of a required signal to interference plus noise ratios (SINR. We first consider the case of a pair of channels, and develop optimum-achieving power control solutions. Then, for the case of multiple channels, we formulate joint duplex model selection, power control, and channel allocation as a mixed integer nonlinear problem (MINLP, and propose an iterative framework to solve it. The proposed iterative framework consists of a duplex mode selection scheme, a near-optimal distributed power control algorithm, and a greedy channel allocation algorithm. We prove the convergence of the proposed iterative framework as well as a lower bound for the greedy channel allocation algorithm. Numerical results show that the proposed schemes effectively improve the sum rate of FDCFNs.

  19. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples.

    Science.gov (United States)

    Mirnejad, Reza; Mohamadi, Mozafar; Piranfar, Vahbeh; Mortazavi, Seied Mojtaba; Kachuei, Reza

    2013-06-01

    To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  20. Long-Range Ordering of Blunt-Ended DNA Tiles on Supported Lipid Bilayers.

    Science.gov (United States)

    Avakyan, Nicole; Conway, Justin W; Sleiman, Hanadi F

    2017-08-30

    Long-range ordering of DNA crossover tiles with blunt ends on lipid bilayers is investigated using atomic force microscopy. "Blunt-ended" tiles do not have single-stranded complementary ends, and thus instead of assembling via base-pairing, they can interact by π-stacking of their duplex ends. This work demonstrates that the balance of base π-stacking interactions between the ends of DNA duplexes, cholesterol-mediated DNA anchoring, and electrostatic DNA binding to supported lipid bilayers (SLBs) presents an opportunity to build dynamic materials with long-range order on a soft support. The tiles are shown to organize into novel tunable surface packing morphologies on the micrometer scale. This work focuses on three-point star (3PS) tiles that are either unmodified or modified with a cholesterol unit and investigates their interactions on supported lipid bilayers. On fluid bilayers, the cholesterol tiles form extended hexagonal arrays with few defects, while the unmodified tiles do not bind. In contrast, both modified and unmodified tiles bind to gel-phase bilayers and produce arrays of new organized morphologies. With increasing tile concentration, we observe a range of motifs, that progressively favor tile-tile packing over duplex-end π-stacking. These structures can selectively pattern domains of phase-separated lipid bilayers, and the patterning is also observed for four-arm cross-tiles. Dynamic blunt end contacts promote error correction and network reconfiguration to maximize favorable interactions with the substrate and are required for the observed tile organization. These results suggest that small blunt-ended tiles can be used as a platform to organize oligonucleotides, nanoparticles, and proteins into extensive networks at the interface with biologically relevant membrane systems or other soft surface materials for applications in cellular recognition, plasmonics, light harvesting, model systems for membrane protein assemblies, or analytical devices.

  1. Full Duplex Prototype of OFDM on GNURadio and USRPs

    OpenAIRE

    Zhou , Wei; Villemaud , Guillaume; Risset , Tanguy

    2014-01-01

    International audience; Full-duplex is a technology in telecommunication domain that can perform transmitting and receiving at the same time and in the same frequency band. The major obstacle of full-duplex is the self-interference (SI). Some previous works have focused on the SI cancellation in radio frequency (RF), and generally on a narrowband signal model. We have implemented a full-duplex prototype with orthogonal frequency-division multiplexing (OFDM) technology on GNURadio and Universa...

  2. Characterization of thermal aging of duplex stainless steel by SQUID

    International Nuclear Information System (INIS)

    Isobe, Y.; Kamimura, A.; Aoki, K.; Nakayasu, F.

    1995-01-01

    Thermal aging is a growing concern for long-term-aged duplex stainless steel piping in nuclear power plants. Superconducting QUantum Interference Device (SQUID) was used for the detection of thermal aging of SUS329 rolled duplex stainless steel and SCS16 cast duplex stainless steel. It was found that the SQUID output signal pattern in the presence of AC magnetic field applied to the specimen was sensitive to the changes in electromagnetic properties due to thermal aging

  3. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

    Science.gov (United States)

    Hendershot, Jenna M.; O'Brien, Patrick J.

    2014-01-01

    Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

  4. The Origins of Microtexture in Duplex Ti Alloys (Preprint)

    National Research Council Canada - National Science Library

    Glavic, M. G; Bartha, B. B; Jha, S. K; Szczepanski, C. J

    2008-01-01

    .... In addition, for a homogeneous duplex microstructures, all of the secondary alpha phase and the majority of the primary alpha grains retain crystallographic coherency, according to the Burgers...

  5. DNA Modified with Metal Nanoparticles: Preparation and Characterization of Ordered Metal-DNA Nanostructures in a Solution and on a Substrate

    Directory of Open Access Journals (Sweden)

    Nina Kasyanenko

    2016-01-01

    Full Text Available DNA interaction with silver and aluminum nanoparticles in a solution has been investigated with the AFM, SEM, dynamic light scattering, viscometry, and spectral methods. The comparison of DNA interaction with nanoparticles synthesized by the reduction of Ag+ ions and with nanoparticles obtained by the electric discharge plasma method was done. DNA metallization in a solution and on n-silicon surface with metal nanoparticles or by the reduction of silver ions after their binding to DNA was executed and studied. It was shown that DNA strands with regular location of silver or aluminum nanoparticles can be prepared. The conditions for the formation of silver nanoparticles and silver nanoclusters on DNA were analyzed.

  6. Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

    Science.gov (United States)

    Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

    2012-01-01

    A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap. PMID:22692744

  7. Development and evaluation of a single-step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gigantica (family Fasciolidae, class Trematoda, phylum Platyhelminthes).

    Science.gov (United States)

    Le, Thanh Hoa; Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

    2012-08-01

    A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

  8. Covalent immobilization of oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for viral DNA sensing.

    Science.gov (United States)

    Ruffien, Audrey; Dequaire, Murielle; Brossier, Pierre

    2003-04-07

    DNA-sensing platforms were prepared by covalently attaching oligonucleotide capture probes onto p-aminophenyl-functionalized carbon surfaces and applied to the determination of an amplified herpes virus DNA sequence in an electrochemical hybridization assay.

  9. Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin. Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves As An Atypical DNA Intercalator

    Science.gov (United States)

    Fekry, Mostafa I.; Szekely, Jozsef; Dutta, Sanjay; Breydo, Leonid; Zang, Hong; Gates, Kent S.

    2012-01-01

    Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M−1. Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allows construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA. PMID:21954957

  10. Full Duplex Emulation via Spatial Separation of Half Duplex Nodes in a Planar Cellular Network

    DEFF Research Database (Denmark)

    Thomsen, Henning; Kim, Dong Min; Popovski, Petar

    2016-01-01

    (HD-BSs), which is a scheme termed CoMPflex (CoMP for In-Band Wireless Full Duplex). A FD-BS can be seen as a special case of CoMPflex with separation distance zero. In this paper we study the performance of CoMPflex in a two-dimensional cellular scenario using stochastic geometry and compare...

  11. Harvesting full-duplex rate gains in cellular networks with half-duplex user terminals

    KAUST Repository

    AlAmmouri, Ahmad

    2016-07-26

    Full-Duplex (FD) transceivers may be expensive in terms of complexity, power consumption, and price to be implemented in all user terminals. Therefore, techniques to exploit in-band full-duplex communication with FD base stations (BSs) and half-duplex (HD) users\\' equipment (UEs) are required. In this context, 3-node topology (3NT) has been recently proposed for FD BSs to reuse the uplink (UL) and downlink (DL) channels with HD terminals within the same cell. In this paper, we present a tractable mathematical framework, based on stochastic geometry, for 3NT in cellular networks. To this end, we propose a design paradigm via pulse-shaping and partial overlap between UL and DL channels to maximize the harvested rate gains in 3NT. The results show that 3NT achieves a close performance to networks with FD BSs and FD UEs, denoted by 2-node topology (2NT) networks. A maximum of 5% rate loss is reported when 3NT is compared to 2NT with efficient self-interference cancellation (SIC). If the SIC in 2NT is not efficient, 3NT highly outperforms 2NT. Consequently, we conclude that, irrespective to the UE duplexing scheme, it is sufficient to have FD BSs to harvest FD rate gains.

  12. Scaffolding along Nucleic Acid Duplexes Using 2'-Amino-Locked Nucleic Acids

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2014-01-01

    depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing...... of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α......-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA...

  13. Detection of chicken contamination in beef meatball using duplex-PCR Cyt b gene

    Science.gov (United States)

    Sari, E. P.; Kartikasari, L. R.; Cahyadi, M.

    2017-04-01

    Beef is one of expensive animal protein sources compared to other meats, on the other hand, chicken is cheap animal protein source. Mixing of chicken into beef meatball is possibly performed to decrease production cost. The aim of this study was to detect chicken contamination in beef meatball using Cytochrome b (Cyt b) gene by duplex-PCR. Sample was designed and prepared as follows, 100% of chicken meatball, 100% of beef meatball and serial level of chicken contaminations in beef meatball (1, 5, 10 and 25%, respectively). Isolation of DNA genome from meatball was according to the guideline of gSYNCTM DNA Extraction Kit for animal tissue. The PCR reaction was carried out using KAPA2G Fast Multiplex Mix. This study found that the DNA genome was succesfully extracted. Moreover, chicken contamination in beef meatball was indicated by the presence of 227 bp DNA band on 2% of agarose gels. Current study revealed that duplex-PCR using Cyt b gene as a genetic marker was able to detect chicken contamination in beef meatball until 1% of chicken meat in the sample. It can be effectively used to identify contamination and also authenticate species origin in animal products to protect consumer from undesirable contents in the food.

  14. Duplex tube steam reformer development program

    Energy Technology Data Exchange (ETDEWEB)

    Lewe, C K; Nieto, J M; Papadopoulos, A

    1978-09-01

    Work done in partial fulfillment of Task 7 of the Duplex Steam Reformer Development Program is described. The DSR concept acts as a double barrier between a process heat high temperature reactor plant (PNP) and a closed loop chemical heat pipe (CHP) for the long distance transport of chemical energy to a remote industrial user. The current state of the DSR design is described as well as related systems and equipment. The PNP concept presented is based upon work currently underway in the Federal Republic of Germany.

  15. Duplex tube steam reformer development program

    International Nuclear Information System (INIS)

    Lewe, C.K.; Nieto, J.M.; Papadopoulos, A.

    1978-09-01

    Work done in partial fulfillment of Task 7 of the Duplex Steam Reformer Development Program is described. The DSR concept acts as a double barrier between a process heat high temperature reactor plant (PNP) and a closed loop chemical heat pipe (CHP) for the long distance transport of chemical energy to a remote industrial user. The current state of the DSR design is described as well as related systems and equipment. The PNP concept presented is based upon work currently underway in the Federal Republic of Germany

  16. Duplex ultrasound: Indications and findings in a newly created ...

    African Journals Online (AJOL)

    Duplex ultrasound: Indications and findings in a newly created facility at the University of Calabar Teaching Hospital, Calabar. ... It is recommended that timely referrals be made, and mobile Doppler units be acquired to save more lives and limbs in the developing world. Keywords: Calabar, deep venous thrombosis, duplex ...

  17. Acoustical and perceptual influence of duplex stringing in grand pianos.

    Science.gov (United States)

    Öberg, Fredrik; Askenfelt, Anders

    2012-01-01

    This study investigates the acoustical and perceptual influence of the string parts outside the speaking length in grand pianos (front and rear duplex strings). Acoustical measurements on a grand piano in concert condition were conducted, measuring the fundamental frequencies of all main and duplex strings in the four octaves D4-C8. Considerable deviations from the nominal harmonic relations between the rear duplex and main string frequencies, as described by the manufacturer in a patent, were observed. Generally the rear duplex strings were tuned higher than the nominal harmonic relations with average and median deviations approaching +50 cent. Single keys reached +190 and -100 cent. The spread in deviation from harmonic relations within trichords was also substantial with average and median values around 25 cent, occasionally reaching 60 cent. Contributions from both front and rear duplex strings were observed in the bridge motion and sound. The audibility of the duplex strings was studied in an ABX listening test. Complete dampening of the front duplex was clearly perceptible both for an experiment group consisting of musicians and a control group with naive subjects. The contribution from the rear duplex could also be perceived, but less pronounced. © 2012 Acoustical Society of America.

  18. Comparison of the voltammetric behavior of metronidazole at a DNA-modified glassy carbon electrode, a mercury thin film electrode and a glassy carbon electrode

    OpenAIRE

    Brett, Ana Maria Oliveira; Serrano, Silvia H. P.; Gutz, Ivano G. R.; La-Scalea, Mauro A.

    1997-01-01

    The electroanalytical performance at three electrodes: DNA-modified galssy carbon electrode, mercury thin film electrode and glassy carbon electrode, for the study of the electrochemical reduction of metronidazole is compared. All three electrodes showed a similar trend in the reduction mechanism for metronidazole, depenent on pH in the acid and neutral region and independent in alkaline media, although there was a shift in the peak potentials to more negative values when a bare glassy carbon...

  19. Study on the wear of TiN/Ti duplex and multilayer coatings in microabrasion tests

    International Nuclear Information System (INIS)

    Flores, M; De Las Heras, E; Ruelas, R; Rodriguez, E; Bautista, A; Pazos, L; Corengia, P

    2008-01-01

    Ionic nitriding, on steels, is used to harden the surface of components improving resistance to corrosion and wear and increasing the stress life. Duplex treatments are an alternative for resolving the limitations of very hard coatings on less hard substrates. In this case the duplex treatment consists of an ionic nitriding treatment followed by a single or multilayer coatings deposited by means of the PVD technique. This work presents the influence of the variation in the severity of contact on the kind of wear present in the microabrasion test used to measure the wear coefficient of duplex coatings, consisting of a layer nitrided by DC-pulsed plasma plus TiN coatings and multilayers of TiN/Ti deposited on non-nitrided and nitrided AISI 316L stainless steel and H13 steel. The severity of contact was modified by varying the charge (0.25 to 1 N). The abrasives used were a suspension of 0.1μm diameter diamond particles and a suspension of an average 5 μm diameter aluminum particles. The influence of the presence of relatively soft metallic layers on the determination of the wear coefficient was analyzed in the metal-ceramic multilayers. Two sphere revolving speeds of de 0.05 and 0.154 m/s were used on two microabrasion machines: one commercial and the other built in the UdeG laboratory. The wear marks were photographed and measured with an optic microscope. The value of the critical charge at which the transition occurs between the methods of wear of the substrates and the nitrited samples was determined. Resistance to the AISI 316L steel's microabrasive wear increases with the ionic nitriding treatment. The duplex coatings increase resistance to the wear from the nitrited samples. The wear resistance of the samples with multilayer coatings surpassed that of the duplex samples with multilayer coatings. The greater resistance of the multilayers may be explained by an increase in the resistance to the fracture and not by a increase in surface hardness. The transition

  20. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    Directory of Open Access Journals (Sweden)

    Svetlana A Perevozchikova

    Full Text Available The DNA mismatch repair (MMR system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

  1. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    Science.gov (United States)

    Perevozchikova, Svetlana A; Trikin, Roman M; Heinze, Roger J; Romanova, Elena A; Oretskaya, Tatiana S; Friedhoff, Peter; Kubareva, Elena A

    2014-01-01

    The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

  2. Selective G-Quadruplex DNA Recognition by a New Class of Designed Cyanines

    Directory of Open Access Journals (Sweden)

    Markus W. Germann

    2013-11-01

    Full Text Available A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

  3. Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

    Science.gov (United States)

    Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenta; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

    2017-10-01

    Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  4. Ethidium bromide modifies the agarose electrophoretic mobility of CAG•CTG alternative DNA structures generated by PCR

    OpenAIRE

    Gomes-Pereira, Mario; Monckton, Darren G.

    2017-01-01

    The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG?CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipp...

  5. Adsorption kinetics and mechanical properties of thiol-modified DNA-oligos on gold investigated by microcantilever sensors

    DEFF Research Database (Denmark)

    Marie, Rodolphe Charly Willy; Jensenius, Henriette; Thaysen, Jacob

    2002-01-01

    Immobilised DNA-oligo layers are scientifically and technologically appealing for a wide range of sensor applications such as DNA chips. Using microcantilever-based sensors with integrated readout, we demonstrate in situ quantitative studies of surface-stress formation during self-assembly of a 25...... on the gold surface after which a significant unspecific adsorption takes place on top of the first DNA-oligo layer. The cantilever-based sensor principle has a wide range of applications in real-time local monitoring of chemical and biological interactions as well as in the detection of specific DNA...

  6. Synthesis of Base-Modified 2 '-Deoxyribonucleoside Triphosphates and Their Use in Enzymatic Synthesis of Modified DNA for Applications in Bioanalysis and Chemical Biology

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal

    2014-01-01

    Roč. 79, č. 21 (2014), s. 9914-9921 ISSN 0022-3263 R&D Projects: GA ČR GBP206/12/G151; GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : cross-coupling reactions * modified nucleoside triphosphates * nucleic acids Subject RIV: CC - Organic Chemistry Impact factor: 4.721, year: 2014

  7. Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands

    Science.gov (United States)

    Talhaoui, Ibtissam; Couve, Sophie; Gros, Laurent; Ishchenko, Alexander A.; Matkarimov, Bakhyt; Saparbaev, Murat K.

    2014-01-01

    The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N6-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes. PMID:24692658

  8. Chronic consumption of a western diet modifies the DNA methylation profile in the frontal cortex of mice.

    Science.gov (United States)

    Yokoyama, Amy S; Dunaway, Keith; Rutkowsky, Jennifer; Rutledge, John C; Milenkovic, Dragan

    2018-02-21

    In our previous work in mice, we have shown that chronic consumption of a Western diet (WD; 42% kcal fat, 0.2% total cholesterol and 34% sucrose) is correlated with impaired cognitive function. Cognitive decline has also been associated with alterations in DNA methylation. Additionally, although there have been many studies analyzing the effect of maternal consumption of a WD on DNA methylation in the offspring, few studies have analyzed how an individual's consumption of a WD can impact his/her DNA methylation. Since the frontal cortex is involved in the regulation of cognitive function and is often affected in cases of cognitive decline, this study aimed to examine how chronic consumption of a WD affects DNA methylation in the frontal cortex of mice. Eight-week-old male mice were fed either a control diet (CD) or a WD for 12 weeks, after which time alterations in DNA methylation were analyzed. Assessment of global DNA methylation in the frontal cortex using dot blot analysis revealed that there was a decrease in global DNA methylation in the WD-fed mice compared with the CD-fed mice. Bioinformatic analysis identified several networks and pathways containing genes displaying differential methylation, particularly those involved in metabolism, cell adhesion and cytoskeleton integrity, inflammation and neurological function. In conclusion, the results from this study suggest that consumption of a WD alters DNA methylation in the frontal cortex of mice and could provide one of the mechanisms by which consumption of a WD impairs cognitive function.

  9. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xianggang [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Cheng Ziqiang, E-mail: czqsd@126.com [College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong (China); Fan Hai [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Ai Shiyun, E-mail: ashy@sdau.edu.cn [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Han Ruixia [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China)

    2011-07-15

    Highlights: > A sensitive electrochemical biosensor for the detection of gene sequence was developed. > The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. > The hybrid nanomaterials could provide a porous structure with good properties. > The biosensor has highly selectivity and sensitivity. > The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10{sup -12} to 1.0 x 10{sup -9} M (R = 0.9863) with a detection limit of 4.3 x 10{sup -13} M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

  10. Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR

    Directory of Open Access Journals (Sweden)

    Mário Gomes-Pereira

    2017-05-01

    Full Text Available The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

  11. Rietveld and impedance analysis of cold and hot rolled duplex and lean duplex steels for application in paper and pulp industry

    International Nuclear Information System (INIS)

    Esteves, Luiza; Lins, Vanessa de Freitas Cunha; Viana, Adolfo Kalergis do Nascimento

    2017-01-01

    In this study, X-Ray Diffraction (XRD) and Rietveld Refinement were performed to identify and quantify the ferrite and austenite phase of cold and hot rolled duplex stainless steels (UNS S31803) and lean duplex stainless steels (UNS S32304). Electrochemical impedance spectroscopy (EIS) was applied to evaluate the chemical behavior of duplex and lean duplex stainless steels in white, green, and black liquors of paper and pulp industry. Rietveld analysis results showed a higher austenite content than the standard limit for duplex steels in the hot rolled condition. The hot rolling condition plays a major role in improving corrosion resistance in white liquor mainly for the lean duplex steel. (author)

  12. Rietveld and impedance analysis of cold and hot rolled duplex and lean duplex steels for application in paper and pulp industry

    Energy Technology Data Exchange (ETDEWEB)

    Esteves, Luiza; Lins, Vanessa de Freitas Cunha, E-mail: luizaeq@yahoo.com.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Engenharia Quimica; Paiva, Paulo Renato Perdigao [Centro Federal de Educacao Tecnologica de Minas Gerais (CEFET), Belo Horizonte, MG (Brazil); Viana, Adolfo Kalergis do Nascimento [APERAM South America, Timoteo, MG (Brazil)

    2017-01-15

    In this study, X-Ray Diffraction (XRD) and Rietveld Refinement were performed to identify and quantify the ferrite and austenite phase of cold and hot rolled duplex stainless steels (UNS S31803) and lean duplex stainless steels (UNS S32304). Electrochemical impedance spectroscopy (EIS) was applied to evaluate the chemical behavior of duplex and lean duplex stainless steels in white, green, and black liquors of paper and pulp industry. Rietveld analysis results showed a higher austenite content than the standard limit for duplex steels in the hot rolled condition. The hot rolling condition plays a major role in improving corrosion resistance in white liquor mainly for the lean duplex steel. (author)

  13. Buffer management in wireless full-duplex systems

    KAUST Repository

    Bouacida, Nader

    2015-10-19

    Wireless full-duplex radios can simultaneously transmit and receive using the same frequency. In theory, this can double the throughput. In fact, there is only little work addressing aspects other than throughput gains in full-duplex systems. Over-buffering in today\\'s networks or the so-called “bufferbloat” phenomenon creates excessive end-to-end delays resulting in network performance degradation. Our analysis shows that full-duplex systems may suffer from high latency caused by bloated buffers. In this paper, we address the problem of buffer management in full-duplex networks by using Wireless Queue Management (WQM), which is an active queue management technique for wireless networks. Our solution is based on Relay Full-Duplex MAC (RFD-MAC), an asynchronous media access control protocol designed for relay full-duplexing. We compare the performance of WQM in full-duplex environment to Drop Tail mechanism over various scenarios. Our solution reduces the end-to-end delay by two orders of magnitude while achieving similar throughput in most of the cases.

  14. Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species.

    Science.gov (United States)

    Xia, Qian-Feng; Liu, Jin-Bo; Liu, Ping; Qin, Xi; Qian, Shi-Yun; Tu, Zhi-Guang

    2012-01-01

    We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

  15. Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

    Science.gov (United States)

    Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Secondary Hardening Behavior in Super Duplex Stainless Steels during LCF in Dynamic Strain Ageing Regime

    OpenAIRE

    Chai, Guocai; Andersson, Marcus

    2013-01-01

    Cyclic deformation behaviors in five modified duplex stainless steel S32705 grades have been studied at 20 °C, 200 °C, 250° and 350 °C. The influence of temperature and nitrogen concentration on the occurrence of the second hardening phenomenon, in the stress response curve was focused. An increase in nitrogen concentration can have a positive effect on dynamic strain ageing by increasing the first hardening and also the second hardening behavior during cyclic deformation. Furthermore, an inc...

  17. Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

    DEFF Research Database (Denmark)

    Ries, Annika; Kumar, Rajesh; Lou, Chenguang

    2016-01-01

    Galactose-modified thymidine, LNA-T and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2...

  18. Duplex ultrasound for identifying renal artery stenosis

    DEFF Research Database (Denmark)

    Zachrisson, Karin; Herlitz, Hans; Lönn, Lars

    2017-01-01

    Background Renal artery duplex ultrasound (RADUS) is an established method for diagnosis of renal artery stenosis (RAS), but there is no consensus regarding optimal RADUS criteria. Purpose To define optimal cutoff values for RADUS parameters when screening for RAS using intra-arterial trans......-stenotic pressure gradient measurement (PGM) as reference. Material and Methods The renal arteries of 58 consecutive patients evaluated for renovascular hypertension were examined by RADUS and PGM. Conclusive measurements with both methods were obtained in 76 arteries. Hemodynamically significant RAS was defined...... as PGM ≥15 mmHg and was found in 43 of the 76 arteries. RADUS parameters included renal artery peak systolic velocity (PSV) and the renal-aortic ratio (RAR) of flow velocities. Receiver operating characteristic curves (ROCs) and Youden's index were used to calculate optimal RADUS criteria for RAS...

  19. Selection of Suitable DNA Extraction Methods for Genetically Modified Maize 3272, and Development and Evaluation of an Event-Specific Quantitative PCR Method for 3272.

    Science.gov (United States)

    Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

    2016-01-01

    A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize, 3272. We first attempted to obtain genome DNA from this maize using a DNeasy Plant Maxi kit and a DNeasy Plant Mini kit, which have been widely utilized in our previous studies, but DNA extraction yields from 3272 were markedly lower than those from non-GM maize seeds. However, lowering of DNA extraction yields was not observed with GM quicker or Genomic-tip 20/G. We chose GM quicker for evaluation of the quantitative method. We prepared a standard plasmid for 3272 quantification. The conversion factor (Cf), which is required to calculate the amount of a genetically modified organism (GMO), was experimentally determined for two real-time PCR instruments, the Applied Biosystems 7900HT (the ABI 7900) and the Applied Biosystems 7500 (the ABI7500). The determined Cf values were 0.60 and 0.59 for the ABI 7900 and the ABI 7500, respectively. To evaluate the developed method, a blind test was conducted as part of an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDr). The determined values were similar to those in our previous validation studies. The limit of quantitation for the method was estimated to be 0.5% or less, and we concluded that the developed method would be suitable and practical for detection and quantification of 3272.

  20. Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers.

    Science.gov (United States)

    Howland, Shanshan W; Poh, Chek-Meng; Rénia, Laurent

    2011-09-01

    Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5'→3' exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Electrochemical behavior of 7-deazaguanine- and 7-deazaadenine-modified DNA at the hanging mercury drop electrode

    Czech Academy of Sciences Publication Activity Database

    Dudová, Zdenka; Špaček, Jan; Tomaško, Martin; Havran, Luděk; Pivoňková, Hana; Fojta, Miroslav

    2016-01-01

    Roč. 148, č. 1 (2016), s. 3-11 ISSN 0026-9247 Institutional support: RVO:68081707 Keywords : 7-Deazaguanine * 7-Deazaadenine * DNA electrochemistry Subject RIV: BO - Biophysics Impact factor: 1.282, year: 2016

  2. Electrochemical monitoring of the interaction between mitomycin C and DNA at chitosan--carbon nanotube composite modified electrodes

    OpenAIRE

    CANAVAR, Pembe Ece; EKŞİN, Ece; ERDEM, Arzum

    2015-01-01

    Single-walled carbon nanotube (CNT) and chitosan composite (chitosan*CNT) based sensors were developed as DNA biosensors, and then they were applied for electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA. The oxidation signals of MC and guanine were monitored before and after the interaction process by differential pulse voltammetry (DPV). The DPV results were in good agreement with those of electrochemical impedance spectroscopy (EIS)....

  3. Use of polished and mercury film-modified silver solid amalgam electrodes in electrochemical analysis of DNA

    Czech Academy of Sciences Publication Activity Database

    Fadrná, Renata; Cahová, Kateřina; Havran, Luděk; Josypčuk, Bohdan; Fojta, Miroslav

    2005-01-01

    Roč. 17, 5-6 (2005), s. 452-459 ISSN 1040-0397 R&D Projects: GA MPO 1H-PK/42; GA ČR GA203/04/1325; GA AV ČR KJB4004302 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z50040507 Keywords : DNA electrochemistry * solid amalgam electrodes * mercury film electrodes * DNA damage Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.189, year: 2005

  4. Charge transport through DNA/DNA duplexes and DNA/RNA hybrids: complex mechanism study

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

    Roč. 20, č. 1 (2013), s. 9-9 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /11./. 14.03.2013-16.03.2013, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378271 ; RVO:86652036 Keywords : charge transport * fluorescence spectroscopy * DFT Subject RIV: CF - Physical ; Theoretical Chemistry

  5. The ABCs of molecular dynamics simulations on B-DNA, circa 2012

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... This article provides a retrospective on the ABC initiative in the area of all-atom molecular dynamics (MD) simulations including explicit solvent on all tetranucleotide steps of duplex B-form DNA duplex, ca. 2012. The ABC consortium has completed two phases of simulations, the most current being a set of ...

  6. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    Science.gov (United States)

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

  7. Randomly detected genetically modified (GM maize (Zea mays L. near a transport route revealed a fragile 45S rDNA phenotype.

    Directory of Open Access Journals (Sweden)

    Nomar Espinosa Waminal

    Full Text Available Monitoring of genetically modified (GM crops has been emphasized to prevent their potential effects on the environment and human health. Monitoring of the inadvertent dispersal of transgenic maize in several fields and transport routes in Korea was carried out by qualitative multiplex PCR, and molecular analyses were conducted to identify the events of the collected GM maize. Cytogenetic investigations through fluorescence in situ hybridization (FISH of the GM maize were performed to check for possible changes in the 45S rDNA cluster because this cluster was reported to be sensitive to replication and transcription stress. Three GM maize kernels were collected from a transport route near Incheon port, Korea, and each was found to contain NK603, stacked MON863 x NK603, and stacked NK603 x MON810 inserts, respectively. Cytogenetic analysis of the GM maize containing the stacked NK603 x MON810 insert revealed two normal compact 5S rDNA signals, but the 45S rDNA showed a fragile phenotype, demonstrating a "beads-on-a-string" fragmentation pattern, which seems to be a consequence of genetic modification. Implications of the 45S rDNA cluster fragility in GM maize are also discussed.

  8. Development of oxide dispersion strengthened 2205 duplex stainless steel composite

    Directory of Open Access Journals (Sweden)

    Oladayo OLANIRAN

    2015-05-01

    Full Text Available Composites of duplex stainless steel were produced by oxide dispersion strengthening with comparatively improved mechanical properties by hot press sintering of partially stabilized Zirconia (PSZ, 3% yttria, mole fraction dispersion in 2205 duplex stainless steels. Ceramic oxide was added as reinforcement, while chromium (Cr and Nickel (Ni were incorporated to maintain the austenitic/ferritic phase balance of the duplex stainless steel. The powders and sintered were characterized in detail using scanning electron microscopy (SEM and X-ray diffraction (XRD. The microstructural evolution and phase formation during oxide dispersion strengthening of duplex stainless steel composites were investigated. The influence of composition variation of the reinforcements on the microstructural and corrosion behaviour in simulated mine water of the composites were investigated. In this manuscript, it was established that composition has great influence on the structure/properties relationship of the composites developed.

  9. Improper Signaling for Virtual Full-Duplex Relay Systems

    KAUST Repository

    Gaafar, Mohamed

    2017-02-14

    Virtual full-duplex (VFD) is a powerful solution to compensate the rate loss of half-duplex relaying without the need to full-duplex capable nodes. Inter-relay interference (IRI) challenges the operation of VFD relaying systems. Recently, improper signaling is employed at both relays of the VFD to mitigate the IRI by imposing the same signal characteristics for both relays. To further boost the achievable rate performance, asymmetric time sharing VFD relaying system is adopted with different improper signals at the half-duplex relays. The joint tuning of the three design parameters improves the achievable rate performance at different ranges of IRI and different relays locations. Extensive simulation results are presented and analyzed to show the achievable rate gain of the proposed system and understand the system behavior.

  10. Atomistic Simulations of Complex DNA DSBs and the Interactions with Ku70/80 Heterodimer

    Science.gov (United States)

    Hu, Shaowen; Cucinotta, Francis A.

    2011-01-01

    Compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex. Binding of Ku drastically reduces the structural disruption and flexibility caused by the complex lesions. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. Binding of Ku promotes the rigidity of DNA duplexes, due to the clamp structure of the inner surface of the rings of Ku70/80.

  11. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic......-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary...

  12. Structure of HIV-1 reverse transcriptase bound to a novel 38-mer hairpin template-primer DNA aptamer.

    Science.gov (United States)

    Miller, Matthew T; Tuske, Steve; Das, Kalyan; DeStefano, Jeffrey J; Arnold, Eddy

    2016-01-01

    The development of a modified DNA aptamer that binds HIV-1 reverse transcriptase (RT) with ultra-high affinity has enabled the X-ray structure determination of an HIV-1 RT-DNA complex to 2.3 Å resolution without the need for an antibody Fab fragment or RT-DNA cross-linking. The 38-mer hairpin-DNA aptamer has a 15 base-pair duplex, a three-deoxythymidine hairpin loop, and a five-nucleotide 5'-overhang. The aptamer binds RT in a template-primer configuration with the 3'-end positioned at the polymerase active site and has 2'-O-methyl modifications at the second and fourth duplex template nucleotides that interact with the p66 fingers and palm subdomains. This structure represents the highest resolution RT-nucleic acid structure to date. The RT-aptamer complex is catalytically active and can serve as a platform for studying fundamental RT mechanisms and for development of anti-HIV inhibitors through fragment screening and other approaches. Additionally, the structure allows for a detailed look at a unique aptamer design and provides the molecular basis for its remarkably high affinity for RT. © 2015 The Protein Society.

  13. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

  14. Genome-wide DNA methylation profiling by modified reduced representation bisulfite sequencing in Brassica rapa suggests that epigenetic modifications play a key role in polyploid genome evolution

    OpenAIRE

    Chen, Xun; Ge, Xianhong; Wang, Jing; Tan, Chen; King, Graham J.; Liu, Kede

    2015-01-01

    Brassica rapa includes some of the most important vegetables worldwide as well as oilseed crops. The complete annotated genome sequence confirmed its paleohexaploid origins and provides opportunities for exploring the detailed process of polyploid genome evolution. We generated a genome-wide DNA methylation profile for B. rapa using a modified reduced representation bisulfite sequencing (RRBS) method. This sampling represented 2.24% of all CG loci (2.5 × 105), 2.16% CHG (2.7 × 105), and 1.68%...

  15. Dynamic Chemistry of Disulfide Terminated Oligonucleotides in Duplexes and Double-Crossover Tiles.

    Science.gov (United States)

    De Stefano, Mattia; Vesterager Gothelf, Kurt

    2016-06-16

    Designed nanostructures formed by self-assembly of multiple DNA strands suffer from low stability at elevated temperature and under other denaturing conditions. Here, we propose a method for covalent coupling of DNA strands in such structures by the formation of disulfide bonds; this allows disassembly of the structure under reducing conditions. The dynamic chemistry of disulfides and thiols was applied to crosslink DNA strands with terminal disulfide modifications. The formation of disulfide-linked DNA duplexes consisting of three strands is demonstrated, as well as a more-complex DNA double-crossover tile. All the strands in the fully disulfide-linked structures are covalently and geometrically interlocked, and it is demonstrated that the structures are stable under heating and in the presence of denaturants. Such a reversible system can be exploited in applications where higher DNA stability is needed only temporarily, such as delivery of cargoes to cells by DNA nanostructures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Magnetic resonance urography in duplex kidney with ectopic ureteral insertion

    Directory of Open Access Journals (Sweden)

    Conjeevaram Rajendrarao Thambidorai

    2011-01-01

    Full Text Available This is a report on the use of magnetic resonance urography (MRU in a 6-year-old girl who presented with urinary incontinence. She had a left duplex kidney with poorly functioning upper moiety and ectopic insertion of the dilated upper pole ureter. MRU has been shown to be superior to conventional imaging techniques in delineating poorly functioning moieties of duplex kidneys and ectopic ureters.

  17. The role of duplex stainless steels for downhole tubulars

    International Nuclear Information System (INIS)

    Francis, R.

    1993-01-01

    In sour conditions there is an increasing trend to turn to corrosion resistant alloys for downhole tubulars. The most commonly used CRA tubular is 13Cr, and there are thousands of feet in service. However, there are limits to the use of 13Cr, ie., the risk of sulphide stress corrosion cracking at high H 2 S levels, and the possibility of pitting or high corrosion rates in waters with high chloride contents. Where the service conditions are felt to be too severe for 13Cr alloys it has been traditional to switch to nickel base alloys such as alloys 825 and C-276 (UNS N08825 and N10276). The alloys are much more expensive than 13Cr, and in recent years the duplex stainless steels have been selected as alloys with superior corrosion and SSCC resistance compared with 13Cr, and having lower cost than nickel alloys. Originally the 22Cr duplex alloy (UNS 31803) was used, but more recently the 25Cr super duplex alloys (UNS S32760 and S32750) have become more available. The present paper reviews the data available for 13Cr and the limits of applicability. Data is also presented for laboratory tests for both the 22Cr and 25Cr super duplex alloys. There is extensive service experience with both 22Cr and 25Cr super duplex in the North Sea, covering both downhole tubulars, manifold and post wellhead equipment. Data is presented showing some of the sour condition being experienced in the North Sea by super duplex alloys. These results show that there is a substantial gap between the limits of use for 13Cr and the 25Cr super duplex stainless steel alloys. This means that in many sour environments super duplex stainless steel provides a cost effective alternative to nickel-base alloys

  18. Duplex colour Doppler sonography — role in neck metastasis

    OpenAIRE

    Puri, R.; Srinath, V. S.; Chakravarti, A. L.

    2002-01-01

    The present study was done to evaluate the rote of Duplex colour Doppler Sonography in neck metastasis. 30 patients with primary head and neck cancer were evaluated by Duplex colour Doppier sonography and results analysed. In more than 95% of cases, the vascular status could be established before any form of therapy was instituted. Compression and infiltration of carotids and internal jugular vein (IJV) could be demonstrated and a structured and dynamic relationship of metastatic nodes to the...

  19. A NEW APPROACH AT SURFACE ENGINEERING; DUPLEX SURFACE TREATMENT

    Directory of Open Access Journals (Sweden)

    Akgün ALSARAN

    2003-01-01

    Full Text Available Duplex surface process was developed to deposite thin ceramic film coating on commonly manufacturing used low alloy steels. Low alloy steel having low strength by this process was first plasma nitrided with purpose enhancing load bearing capacity and then deposited with such ceramic coating. In this study, the duplex surface treatment was deal with main lines and informed about advantage and disadvantage.

  20. Bypass of a protein barrier by a replicative DNA helicase

    NARCIS (Netherlands)

    Yardimci, Hasan; Wang, Xindan; Loveland, Anna B.; Tappin, Inger; Rudner, David Z.; Hurwitz, Jerard; van Oijen, Antoine M.; Walter, Johannes C.

    2012-01-01

    Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is

  1. Timing, Coordination, and Rhythm : Acrobatics at the DNA Replication Fork

    NARCIS (Netherlands)

    Hamdan, Samir M.; Oijen, Antoine M. van

    2010-01-01

    In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In

  2. Surface plasmon resonator using high sensitive resonance telecommunication wavelengths for DNA sensors of Mycobacterium tuberculosis with thiol-modified probes.

    Science.gov (United States)

    Hsu, Shih-Hsiang; Hung, Shao-Chiang; Chen, Yu-Kun; Jian, Zhi-Hao

    2014-12-25

    Various analytes can be verified by surface plasmon resonance, thus continuous improvement of this sensing technology is crucial for better sensing selection and higher sensitivity. The SPR sensitivity on the wavelength modulation is enhanced with increasing wavelengths. The telecommunication wavelength range was then utilized to detect Mycobacterium tuberculosis (MTB) deoxyribonucleic acid (DNA) under two situations, without immobilization and with 5'-thiol end labeled IS6100 DNA probes, for SPR sensitivity comparison. The experimental data demonstrated that the SPR sensitivity increased more than 13 times with the wavelength modulation after immobilization. Since the operating wavelength accuracy of a tunable laser source can be controlled within 0.001 nm, the sensitivity and resolution on immobilized MTB DNA were determined as 1.04 nm/(μg/mL) and 0.9 ng/mL, respectively.

  3. Bacillus subtilis 168 RecR protein-DNA complexes visualized as looped structures.

    Science.gov (United States)

    Ayora, S; Stiege, A C; Lurz, R; Alonso, J C

    1997-03-18

    The Bacillus subtilis 168 RecR protein bound to duplex DNA in the presence of ATP and divalent cations (Mg2+ and Zn2+) was visualized by electron microscopy as a nearly spherical particle. A RecR homomultimer is frequently located at the intersection of two duplex DNA strands in an interwound DNA molecule, generating DNA loops of variable length. Two individual DNA molecules bound to the same protein are seen at a very low frequency, if at all. The association of RecR with the intersection of two duplex DNA strands is more often seen in supercoiled than with relaxed or linear DNA. The RecR protein displays a slight but significant preference for negatively supercoiled over linear DNA. The minimum substrate size for RecR protein is about 150 bp in length. A possible mechanism for RecR function in DNA repair is discussed.

  4. EFFECT OF INTERMETALLIC PHASES ON CORROSION BEHAVIOR AND MECHANICAL PROPERTIES OF DUPLEX STAINLESS STEEL AND SUPER-DUPLEX STAINLESS STEEL

    OpenAIRE

    Prabhu Paulraj; Rajnish Garg

    2015-01-01

    Duplex Stainless Steels (DSS) and Super Duplex Stainless Steel (SDSS) have excellent integration of mechanical and corrosion properties. However, the formation of intermetallic phases is a major problem in their usage. The mechanical and corrosion properties are deteriorated due to the presence of intermetallic phases. These phases are induced during welding, prolonged exposure to high temperatures, and improper heat treatments. The main emphasis of this review article is on intermetallic pha...

  5. Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

    International Nuclear Information System (INIS)

    Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

    1987-01-01

    α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

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

    Science.gov (United States)

    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.

  7. Structure of a DNA glycosylase that unhooks interstrand cross-links

    Energy Technology Data Exchange (ETDEWEB)

    Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)

    2017-04-10

    DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

  8. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates

    Czech Academy of Sciences Publication Activity Database

    Güixens-Gallardo, Pedro; Hocek, Michal; Perlíková, Pavla

    2016-01-01

    Roč. 26, č. 2 (2016), s. 288-291 ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : DNA polymerases * nucleotide addition * primer extension * oligonucleotides * twisted intercalating nucleic acid Subject RIV: CC - Organic Chemistry Impact factor: 2.454, year: 2016

  9. Genetic polymorphisms in DNA repair and oxidative stress pathways may modify the association between body size and postmenopausal breast cancer

    Czech Academy of Sciences Publication Activity Database

    McCullough, L. E.; Eng, S. M.; Bradshaw, P. T.; Cleveland, R. J.; Steck, S. E.; Terry, M. B.; Shen, J.; Crew, K.D.; Rössner ml., Pavel; Ahn, J.; Ambrosone, Ch.B.; Teitelbaum, S. L.; Neugut, A. I.; Santella, R. M.; Gammon, M. D.

    2015-01-01

    Roč. 25, č. 4 (2015), s. 263-269 ISSN 1047-2797 Institutional support: RVO:68378041 Keywords : breast cancer * body mass index * oxidative stress * DNA repair * Epidemiology Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.335, year: 2015

  10. Detection of genetically modified organisms in foods by protein- and DNA-based techniques : bridging the methods

    NARCIS (Netherlands)

    Duijn, G. van; Biert, R. van; Bleeker-Marcelis, H.; Boeijen, I. van; Adan, A.J.; Jhakrie, S.; Hessing, M.

    2002-01-01

    According to European Commission (EC) Regulation 1139/98, foods and food ingredients that are to be delivered to the final consumer in which either protein or DNA resulting from genetic modification is present, shall be subject to additional specific labeling requirements. Since 1994, genetically

  11. Streptavidin-modified monodispersed magnetic poly(2-hydroxyethyl methacrylate) microspheres as solid support in DNA-based molecular protocols

    Czech Academy of Sciences Publication Activity Database

    Salih, T.; Ahlford, A.; Nilsson, M.; Plichta, Zdeněk; Horák, Daniel

    2016-01-01

    Roč. 61, 1 April (2016), s. 362-367 ISSN 0928-4931 R&D Projects: GA MŠk 7E12054 EU Projects: European Commission(XE) 259796 - DIATOOLS Institutional support: RVO:61389013 Keywords : rolling circle amplification * DNA * magnetic microspheres Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.164, year: 2016

  12. Synthesis, micellization behavior, antimicrobial and intercalative DNA binding of some novel surfactant copper(II) complexes containing modified phenanthroline ligands.

    Science.gov (United States)

    Nagaraj, Karuppiah; Ambika, Subramanian; Rajasri, Shanmugasundaram; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-10-01

    The novel surfactant copper(II) complexes, [Cu(ip)2DA](ClO4)21, [Cu(dpqc)2DA](ClO4)22, [Cu(dppn)2DA](ClO4)23, where ip=imidazo[4,5-f][1,10]phenanthroline, dpqc=dipyrido[3,2-a:2',4'-c](6,7,8,9-tetrahydro)phenazine, dppn=benzo[1]dipyrido[3,2-a':2',3'-c]phenazine and DA-dodecylamine, were synthesized and characterized by physico-chemical and spectroscopic methods. In these complexes 1-3, the geometry of copper metal ions was described as square pyramidal. The critical micelle concentration (CMC) value of these surfactant copper(II) complexes in aqueous solution was found out from conductance measurements. Specific conductivity data at different temperatures served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔGm°, ΔHm° and ΔSm°). The binding interaction of these complexes with DNA (calf thymus DNA) in Tris buffer was studied by physico-chemical techniques. In the presence of the DNA UV-vis spectrum of complexes showed red shift of the absorption band along with significant hypochromicity indicating intercalation of our complexes with nucleic acids. Competitive binding study with ethidium bromide (EB) shows that the complexes exhibit the ability to displace the nucleic acid-bound EB indicating that the complexes bind to nucleic acids in strong competition with EB for the intercalative binding site. Observed changes in the circular dichoric spectra of DNA in the presence of surfactant complexes support the strong binding of complexes with DNA. CV results also confirm this mode of binding. Some significant thermodynamic parameters of the binding of the titled complexes to DNA have also been determined. The results reveal that the extent of DNA binding of 3 was greater than that of 1 and 2. The antibacterial and antifungal screening tests of these complexes have shown good results compared to its precursor chloride complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

    Science.gov (United States)

    Dziegielewska, Barbara; Beerman, Terry A; Bianco, Piero R

    2006-09-01

    To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.

  14. New Economical 19Cr Duplex Stainless Steels

    Science.gov (United States)

    Li, Jun; Zhang, Zixing; Chen, Hong; Xiao, Xueshan; Zhao, Junliang; Jiang, Laizhu

    2012-02-01

    New economical duplex stainless steels (DSSs) containing 19Cr-6Mn- xNi-1.0Mo-0.5W-0.5Cu-0.2N ( x = 0.5 to 2.0) were developed, and the microstructure, impact property, and corrosion resistance of the alloys were studied. The ferrite content increases with the solution treatment temperature, but decreases with an increase in nickel. The sigma phase is not found precipitating in the alloys treated with solution from 1023 K to 1523 K (750 °C to 1250 °C). The low-temperature impact energy of the experimental alloys increases first and then decreases rapidly with an increase in nickel, which is mainly due to the martensite transformation with an increase in austenite. The alloys have a better mechanical property and pitting corrosion resistance than AISI 304. Among the designed DSS alloys, 19Cr-6Mn-1.3Ni-1.0Mo-0.5W-0.5Cu-0.2N is found to be an optimum alloy with proper phase proportion, a better combination of mechanical strength and elongation, and higher pitting corrosion resistance compared with those of the other alloys.

  15. Corrosion behaviour of sintered duplex stainless steels

    Energy Technology Data Exchange (ETDEWEB)

    Utrilla, M. Victoria; Urena, Alejandro; Otero, Enrique; Munez, Claudio Jose [Escuela Superior de Ciencias Experimentales y Tecnologia, Universidad Rey Juan Carlos, C/ Tulipan s/n, 28933 Mostoles, Madrid (Spain)

    2004-07-01

    Duplex austenite-ferrite stainless steels were prepared by mixing austenitic (316L) and ferritic (434L) atomized powders. Although different 316L/434L ratios were prepared, present work centred its study on 50% ferrite - 50% austenite sintered steel. The powders were mixed and pressed at 700 MPa and sintered at 1250 deg. C for 30 min in vacuum. The cooling rate was 5 deg. C/min. Solution treatment was carried out to homogenize the microstructure at 1100 deg. C during 20 min. A microstructural study of the material in solution was performed, evaluating the microstructure, proportion and shape of porosity, and ferrite percentage. This last was measured by two methods, quantitative metallography and Fischer ferrito-metry. The materials were heat treated in the range of 700 to 1000 deg. C, for 10, 30 and 60 min and water quenched, to study the microstructural changes and the influence on the intergranular corrosion resistance. The method used to evaluate the sensitization to the intergranular corrosion was the electrochemical potentio-kinetic reactivation procedure (EPR). The test solution was 0.5 M H{sub 2}SO{sub 4} + 0,01 M KSCN at 30 deg. C. The criterion used to evaluate the sensitization was the ratio between the maximum reactivation density (Ir) and the maximum activation density (Ia). The results of the electrochemical tests were discussed in relation with the microstructures observed at the different heat treatments. (authors)

  16. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes......) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6...... relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only...

  17. Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Liang, Min; Zhang, Chaofan; Zhao, Xiaoxu; He, Qiufen; Cui, Limei; Liu, Xiaoling; Sun, Yan-Hong; Fu, Qun; Ji, Yanchun; Bai, Yidong; Huang, Taosheng; Guan, Min-Xin

    2016-08-15

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρ o cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

    International Nuclear Information System (INIS)

    Wang, Jing; Tochio, Naoya; Takeuchi, Aya; Uewaki, Jun-ichi; Kobayashi, Naohiro; Tate, Shin-ichi

    2013-01-01

    Highlights: •The structure of the oxidized A-domain of human HMGB1 was solved. •Phe38 ring was flipped in the oxidized structure from that in the reduced form. •The flipped ring disables the intercalation into the cisplatinated lesions. •The functionally relevant redox-dependent structural change was described. -- Abstract: HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA

  19. Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara

    International Nuclear Information System (INIS)

    Sharpe, Sally; Hanke, Tomas; Tinsley-Bown, Anne; Dennis, Mike; Dowall, Stuart; McMichael, Andrew; Cranage, Martin

    2003-01-01

    Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 μm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01 + rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals

  20. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

    Science.gov (United States)

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

    2016-01-21

    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Genome-wide DNA Methylation Profiling by Modified Reduced Representation Bisulfite Sequencing in Brassica rapa Suggests that Epigenetic Modifications Play a Key Role in Polyploid Genome Evolution

    Directory of Open Access Journals (Sweden)

    Xun eChen

    2015-10-01

    Full Text Available Brassica rapa includes some of the most important vegetables worldwide as well as oilseed crops. The complete annotated genome sequence confirmed its paleohexaploid origins and provides opportunities for exploring the detailed process of polyploid genome evolution. We generated a genome-wide DNA methylation profile for B. rapa using a modified reduced representation bisulfite sequencing (RRBS method. This sampling represented 2.24% of all CG loci (2.5 x 105, 2.16% CHG (2.7 x 105 and 1.68% CHH loci (1.05 x 105 (where H = A, T or C. Our sampling of DNA methylation in B. rapa indicated that 52.4% of CG sites were present as 5mCG, with 31.8% of CHG and 8.3% of CHH. It was found that genic regions of single copy genes had significantly higher methylation compared to those of two or three copy genes. Differences in degree of genic DNA methylation were observed in a hierarchical relationship corresponding to the relative age of the three ancestral subgenomes, primarily accounted by single-copy genes. RNA-seq analysis revealed that overall the level of transcription was negatively correlated with mean gene methylation content and depended on copy number or associated with the different subgenomes. These results provide new insights into the role epigenetic variation plays in polyploid genome evolution, and suggest an alternative mechanism for duplicate gene loss.

  2. Genome-wide DNA methylation profiling by modified reduced representation bisulfite sequencing in Brassica rapa suggests that epigenetic modifications play a key role in polyploid genome evolution.

    Science.gov (United States)

    Chen, Xun; Ge, Xianhong; Wang, Jing; Tan, Chen; King, Graham J; Liu, Kede

    2015-01-01

    Brassica rapa includes some of the most important vegetables worldwide as well as oilseed crops. The complete annotated genome sequence confirmed its paleohexaploid origins and provides opportunities for exploring the detailed process of polyploid genome evolution. We generated a genome-wide DNA methylation profile for B. rapa using a modified reduced representation bisulfite sequencing (RRBS) method. This sampling represented 2.24% of all CG loci (2.5 × 10(5)), 2.16% CHG (2.7 × 10(5)), and 1.68% CHH loci (1.05 × 10(5)) (where H = A, T, or C). Our sampling of DNA methylation in B. rapa indicated that 52.4% of CG sites were present as (5m)CG, with 31.8% of CHG and 8.3% of CHH. It was found that genic regions of single copy genes had significantly higher methylation compared to those of two or three copy genes. Differences in degree of genic DNA methylation were observed in a hierarchical relationship corresponding to the relative age of the three ancestral subgenomes, primarily accounted by single-copy genes. RNA-seq analysis revealed that overall the level of transcription was negatively correlated with mean gene methylation content and depended on copy number or was associated with the different subgenomes. These results provide new insights into the role epigenetic variation plays in polyploid genome evolution, and suggest an alternative mechanism for duplicate gene loss.

  3. Atomic Simulation of Complex DNA DSBs and the Interactions with the Ku70/80 Heterodimer

    Science.gov (United States)

    Hu, Shaowen; Cucinotta, Francis A.

    2011-01-01

    DNA double strand breaks (DSBs) induced by ionizing radiation (IR) usually contain modified bases such as 8-oxo-7,8-dihydroguanine (8-oxoG) and thymine glycol, apurinic/apyrimidinic (AP) sites, 2-deoxyribonolactone, or single-strand breaks (SSBs). The presence of such lesions in close proximity to the DSB terminus makes the DNA nicks more difficult to repair and rejoin than endogenously induced simple DSBs, and as such a major determinant of the biological effects of high linear energy transfer (LET) radiation as encountered in space travel. In this study we conducted molecular dynamics simulations on a series of DNA duplexes with various complex lesions of 8-oxoG and AP sites, in an effort to investigate the effects of such lesions to the structural integrity and stability of DNA after insulted by IR. We also simulated the interaction of such complex DSBs with the Ku70/80 heterodimer, the first protein in mammalian cells to embark the non-homologous end joining (NHEJ) DNA repair pathway. The results indicate, compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex, thus they present more deleterious effects to the genome integrity if not captured and repaired promptly in cells. Simulations also demonstrate the binding of Ku drastically reduces structural disruption and flexibility caused by the complex lesions, and the interactions of Ku with complex DSBs have a different potential energy landscape from the bound structure with simple DSB. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. This energy shift may help the Ku protein to secure at the DSB terminus for a longer time, so that other end processing factors or repair pathways can proceed at the lesions before NHEJ repair process starts. These atomic simulations may provide valuable new insight into the

  4. Mild hydration of didecyldimethylammonium chloride modified DNA by 1H-nuclear magnetic resonance and by sorption isotherm

    Science.gov (United States)

    Harańczyk, H.; Kobierski, J.; Nizioł, J.; Hebda, E.; Pielichowski, J.; Zalitacz, D.; Marzec, M.; El-Ghayoury, A.

    2013-01-01

    The gaseous phase hydration of deoxyribonucleic acid and didecyldimethylammonium chloride (C19H42ClN) complexes (DNA-DDCA) was observed using hydration kinetics, sorption isotherm, and high power nuclear magnetic resonance. Three bound water fractions were distinguished: (i) a very tightly bound water not removed by incubation over silica gel, (ii) a tightly bound water saturating with the hydration time t1h = (0.59 ± 0.04) h, and a loosely bound water fraction, (iii) with the hydration time t2h = (20.9 ± 1.3) h. Proton free induction decay was decomposed into the signal associated with the solid matrix of DNA-DDCA complex (T2S∗≈ 30 μs) and two liquid signal components coming from tightly bound (T2L1∗≈ 100 μs) and from loosely bound water fraction (T2L2∗≈ 1000 μs).

  5. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Simonova, Anna; Bialek-Pietras, M.; Olejniczak, A.; Lesnikowski, Z. J.; Hocek, Michal

    2017-01-01

    Roč. 27, č. 21 (2017), s. 4786-4788 ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : nucleotides * nucleoside triphosphates * carboranes * DNA polymerase * oligonucleotides Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.454, year: 2016

  6. Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

    Science.gov (United States)

    Mitchell, Jonathan K.; Byers, Nathaniel M.

    2013-01-01

    The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein. PMID:24027328

  7. Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

    Directory of Open Access Journals (Sweden)

    Na Ying

    Full Text Available MicroRNAs (miRNAs constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN and hybridization chain reaction (HCR. A reporter probe (RP, comprising recognition sequence (3' end modified with biotin for a target miRNA of miR-21 and capture sequence (5' end modified with Fam for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP, the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio. A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA coated magnetic bead (MB. The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3 in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

  8. Efficacy of duplex ultrasound surveillance after infrainguinal vein bypass may be enhanced by identification of characteristics predictive of graft stenosis development.

    Science.gov (United States)

    Tinder, Chelsey N; Chavanpun, Joe P; Bandyk, Dennis F; Armstrong, Paul A; Back, Martin R; Johnson, Brad L; Shames, Murray L

    2008-09-01

    (15%) limbs, the bypass graft failed and 20 (6%) limbs required amputation. The efficacy of duplex surveillance after infrainguinal vein bypass may be enhanced by modifying testing protocols, eg, rigorous surveillance for "higher risk" bypasses, based on the initial duplex scan results and other characteristics (warfarin therapy, non- single segment saphenous vein conduit, redo bypass) predictive for stenosis development.

  9. Effects of Solution Annealing Temperature on the Galvanic Corrosion Behavior of the Super Duplex Stainless Steels

    Science.gov (United States)

    Lee, Jun-Seob; Jeon, Soon-Hyeok; Park, Yong-Soo

    2013-02-01

    This study investigated the active dissolution of super duplex stainless steel (SDSS) at various solution annealing temperatures. The active dissolutions of the α-phase and γ-phase were compared, and the effects of the surface area ratio on the active dissolutions of both phases were investigated. There were two peaks in the active-passive transition region in the potentiodynamic test in the modified green-death solution. The two peaks changed as the solution annealing temperature was increased from 1050 to 1150 °C. The solution annealing temperature difference affected the critical anodic current densities. This provides useful information for determining the appropriate solution annealing temperature in the modified green-death solution for SDSS.

  10. Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNA

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Nes, Ingolf F.; Wells, Robert D.

    1976-01-01

    Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100–200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced...

  11. Normalizing cDNA libraries.

    Science.gov (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  12. Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food.

    Science.gov (United States)

    Brodmann, Peter D; Ilg, Evelyn C; Berthoud, Hélène; Herrmann, Andre

    2002-01-01

    Quantitative detection methods are needed for enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling threshold, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are approved in the European Union: the insect-resistant Bt176 maize (Maximizer), Btl 1 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymerase chain reaction detection methods were developed for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system.

  13. Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Frauke Hausburg

    2015-02-01

    Full Text Available Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC and pseudouridine-5'-triphosphate (Ψ. We then investigated their effect on i protein expression efficiencies and ii cell viability for human mesenchymal stem cells (hMSCs and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

  14. Four highly pseudosymmetric and/or twinned structures of d(CGCGCG) 2 extend the repertoire of crystal structures of Z-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Zhipu; Dauter, Zbigniew; Gilski, Miroslaw

    2017-10-30

    DNA oligomer duplexes containing alternating cytosines and guanines in their sequences tend to form left-handed helices of the Z-DNA type, with the sugar and phosphate backbone in a zigzag conformation and a helical repeat of two successive nucleotides. Z-DNA duplexes usually crystallize as hexagonally arranged parallel helical tubes, with various relative orientations and translation of neighboring duplexes. Four novel high-resolution crystal structures of d(CGCGCG)2duplexes are described here. They are characterized by a high degree of pseudosymmetry and/or twinning, with three or four independent duplexes differently oriented in a monoclinicP21lattice of hexagonal metric. The various twinning criteria give somewhat conflicting indications in these complicated cases of crystal pathology. The details of molecular packing in these crystal structures are compared with other known crystal forms of Z-DNA.

  15. and OMeN-modified oligonucleotide probes on the stability and ...

    Indian Academy of Sciences (India)

    Melting temperatures (°C) of duplexes containing a DNA:DNA mismatch ... The lower cases and upper cases represent LNA and DNA nucleotides, respectively. ... Forward primer 6. Forward primer 7. Forward primer 8. 5' TTCTC AAGGA CCACC GCATC. 5' TTCTC AAGGA CCACC GCATc. 5' TTCTC AAGGA CCACC ...

  16. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

    Science.gov (United States)

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela

    2015-06-01

    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.

  17. A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.

    Science.gov (United States)

    Venczel, R; Knoke, L; Pavlovic, M; Dzaferovic, E; Vaculova, T; Silaghi, C; Overzier, E; Konrad, R; Kolenčík, S; Derdakova, M; Sing, A; Schaub, G A; Margos, G; Fingerle, V

    2016-02-01

    For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

  18. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2016-09-30

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Characterization of a DNA damage-recognition protein mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

    International Nuclear Information System (INIS)

    Donahue, B.A.; Augot, M.; Bellon, S.F.; Treiber, D.K.; Toney, J.H.; Lippard, S.J.; Essigmann, J.M.

    1990-01-01

    A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, K d , of the protein from cisplatin-modified DNA was estimated to be (1-20) x 10 -10 M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl 2 ] (en, ethylenediamine), and [Pt(dach)Cl 2 ] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl 2 , CdCl 2 , CoCl 2 , or ZnCl 2 and with 1 mM HgCl 2 . This protein, alone or in conjunction with other cellular constituents, could be of general importance in the initial stages of processing of mammalian DNA damaged by cisplatin or other genotoxic agents and may belong to a wider class of such cellular damage-recognition proteins (DRPs)

  2. Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease

    Science.gov (United States)

    Medici, Valentina; Shibata, Noreene M; Kharbanda, Kusum K; Islam, Mohammad S; Keen, Carl L; Kim, Kyoungmi; Tillman, Brittany; French, Samuel W; Halsted, Charles H; LaSalle, Janine M

    2014-01-01

    Maternal diet can affect fetal gene expression through epigenetic mechanisms. Wilson disease (WD), which is caused by autosomal recessive mutations in ATP7B encoding a biliary copper transporter, is characterized by excessive hepatic copper accumulation, but variability in disease severity. We tested the hypothesis that gestational supply of dietary methyl groups modifies fetal DNA methylation and expression of genes involved in methionine and lipid metabolism that are impaired prior to hepatic steatosis in the toxic milk (tx-j) mouse model of WD. Female C3H control and tx-j mice were fed control (choline 8 mmol/Kg of diet) or choline-supplemented (choline 36 mmol/Kg of diet) diets for 2 weeks throughout mating and pregnancy to gestation day 17. A second group of C3H females, half of which were used to cross foster tx-j pups, received the same diet treatments that extended during lactation to 21 d postpartum. Compared with C3H, fetal tx-j livers had significantly lower copper concentrations and significantly lower transcript levels of Cyclin D1 and genes related to methionine and lipid metabolism. Maternal choline supplementation prevented the transcriptional deficits in fetal tx-j liver for multiple genes related to cell growth and metabolism. Global DNA methylation was increased by 17% in tx-j fetal livers after maternal choline treatment (P < 0.05). Maternal dietary choline rescued the lower body weight of 21 d tx-j mice. Our results suggest that WD pathogenesis is modified by maternal in utero factors, including dietary choline. PMID:24220304

  3. Conductivity of natural and modified DNA measured by scanning tunneling microscopy. The effect of sequence, charge and stacking

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, Irena; Král, Karel; Bunček, M.; Víšková, A.; Nešpůrek, Stanislav; Kochalska, Anna; Todorciuc, Tatiana; Weiter, M.; Schneider, Bohdan

    2008-01-01

    Roč. 138, č. 1-2 (2008), s. 3-10 ISSN 0301-4622 R&D Projects: GA ČR GA203/08/1594; GA AV ČR KAN401770651; GA MŠk OC 137; GA AV ČR KAN200100801; GA ČR GA202/07/0643 Grant - others:Marie-Curie RTN BIMORE(XE) MRTN-CT-2006-035859 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40500505; CEZ:AV0Z50520701 Keywords : DNA conductivity * charge transport in molecular systems * STM * electronic properties Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 2.362, year: 2008

  4. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  5. Mechanical properties of welded joints of duplex steels

    International Nuclear Information System (INIS)

    Kawiak, M.; Nowacki, J.

    2003-01-01

    The paper presents the study results of mechanical properties of duplex steels UNS S31803 welded joints as well as duplex and NV A36 steels welded joints. They have ben welded by FCAW method in CO 2 using FCW 2205-H flux-cored wire. The joints have been subjected: tensile tests, impact tests, bending tests, hardness tests and metallographic investigations. The influence of welding parameters and mechanical properties of the joints was appreciated. The welding method assured high tensile strength of the joints (approximately 770 MPa) and high impact strength of the welds (approximately 770 J). All samples were broken outside of welds. (author)

  6. Comparison of simulated and measured quantities of a duplex reactor

    Energy Technology Data Exchange (ETDEWEB)

    Koskela, M.; Kajava, M. [ABB Marine, Helsinki (Finland)

    1997-12-31

    The purpose of this article is to illustrate the use of an analog simulator as a design tool when designing new power electric equipment. The purpose of simulation is to predict the functionality of electrical equipment to be constructed. Duplex reactor is an electromagnetic device designed to reduce voltage harmonics and short circuit currents in the ship electrical network. In this report a comparison between simulated and measured electrical quantities of a duplex reactor has been made. The purpose of the measurements was to show the correct functioning of the reactor. The simulation results and the measured waveforms corresponds well to each other. (orig.) 4 refs.

  7. Corrosion behavior of duplex polyaniline/epoxy coating on mild steel in 3% NaCl

    Directory of Open Access Journals (Sweden)

    Gvozdenović Milica M.

    2005-01-01

    Full Text Available The corrosion behavior and thermal stability of epoxy coatings electrodeposited on mild steel and on mild steel with electrochemically deposited polyaniline (PANI film were investigated by electrochemical impedance spectroscopy (EIS and thermo gravimetric analysis (TGA. The aim of the paper was to present new findings on the corrosion protection of mild steel by a duplex PANI/-epoxy coating in 3% NaCI solution and to determine the effect of thin PANI film on the protective properties of the coating. PANI film was deposited electrochemically on mild steel from an aqueous solution of 0.5 mol dm"3 sodium benzoate and 0.1 mol dm"3 aniline at a constant current density of 1.5 mA cm"2. Non-pigmented epoxy coatings on mild steel and on mild steel with PANI film were obtained by cathodic electrode position at constant voltage and stirring conditions. The resin concentration in the electrode position bath was 10 wt.% solid dispersion in water at pH 5.7. The applied voltage was 250 V, the temperature 26°C and the deposition time 3 min. It was shown that thin PANI film could be used to modify the surface of mild steel prior to epoxy coating deposition, due to the increased corrosion protection of a duplex PANI/epoxy coating comparing to an epoxy coating on mild steel in 3% NaCl solution.

  8. Validation of eDNA markers for New Zealand mudsnail surveillance and initial eDNA monitoring at Mississippi River Basin sites

    Science.gov (United States)

    Merkes, Christopher; Turnquist, Keith N.; Rees, Christopher B.; Amberg, Jon J.

    2015-01-01

    The performance of newly developed New Zealand mudsnail (Potamopyrgus antipodarum; NZMS) genetic markers for environmental (eDNA) analysis of water were compared across two laboratories. The genetic markers were tested in four quantitative polymerase chain reaction assays targeting two regions of the NZMS mitochondrial genome, specifically the cytochrome c oxidase subunit 1 (coi) and cytochrome b (cytb) genes. In a blind study, analysts tested each sample eight times with each assay. There were 10 expected-negative samples from the Black River in La Crosse, Wisconsin, 10 expected-positive samples from the Black Earth Creek in Black Earth, Wisconsin, and 10 known-positive samples from the Black River spiked with NZMS DNA. Previously extracted samples, kept at the Upper Midwest Environmental Sciences Center, were pooled by sample location and then equal quantities were distributed between the Upper Midwest Environmental Sciences Center and the Molecular Conservation Genetics Laboratory at the University of Wisconsin-Stevens Point for analysis. The assays tested were (1) the assay targeting cytb with a minor groove binder probe described by Goldberg and others (2013), (2) the cytb assay with a modified double-quenched probe, (3) an assay targeting coi with a double-quenched probe, and (4) a duplex reaction combining the modified cytb assay and the coi assay. Samples were considered positive for the presence of NZMS DNA when quantitative polymerase chain reaction amplification and probe signal was higher than the normalized threshold value above baseline fluorescence. For the duplex assay, samples were considered positive only when both probe signals were higher than the normalized threshold value above baseline fluorescence. Positive results were then confirmed by sequencing the products.

  9. Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins

    Directory of Open Access Journals (Sweden)

    Sailaja Gangadhara

    2017-12-01

    Full Text Available Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1. To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP, and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env. DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN-γ, and spleen cells producing interleukin (IL-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

  10. Evalution of DNA extraction methods in order to monitor genetically modified materials in soy foodstuffs and feeds commercialised in Turkey by multiplex real-time PCR.

    Science.gov (United States)

    Turkec, Aydin; Kazan, Hande; Baykut, Aykut; Lucas, Stuart J

    2015-01-01

    Soybean is one of the most important biotech crops, widely used as an ingredient in both foodstuffs and feed. DNA extraction methods have been evaluated to detect the presence of genetically modified (GM) materials in soya-containing food and feed products commercialised in Turkey. All extraction methods performed well for the majority of soya foods and feed products analysed. However, the most successful method varied between different products; the Foodproof, Genespin and the cetyltrimethylammonium bromide (CTAB) methods each produced the highest DNA yield and purity for different soya foodstuffs and feeds. Of the samples tested, 20% were positive for the presence of at least two GM elements (35S/NOS) while 11% contained an additional GM element (35S/NOS/FMV). Of the tested products, animal feeds showed a larger prevalence of GM material (50%) than the soya-containing foodstuffs (13%). The best performing extraction methods proved to be the Foodproof, Genespin and CTAB methods for soya-containing food and feed products. The results obtained herein clearly demonstrate the presence of GM soybean in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of soy-containing food and feed products. © 2014 Society of Chemical Industry.

  11. Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells.

    Science.gov (United States)

    Schott, Sarah; Niessner, Heike; Sinnberg, Tobias; Venturelli, Sascha; Berger, Alexander; Ikenberg, Kristian; Villanueva, Jessie; Meier, Friedegund; Garbe, Claus; Busch, Christian

    2012-11-01

    Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5' → 1-O)-2-O-octadecyl-sn-glycerylyl-(3-O → 5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 μM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation. Copyright © 2012 UICC.

  12. Enantiospecific kinking of DNA by a partially intercalating metal complex

    KAUST Repository

    Reymer, Anna

    2012-01-01

    Opposite enantiomers of [Ru(phenanthroline) 3] 2+ affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced by wedge-intercalation of one but not the other enantiomer. This effect is exploited by several classes of DNA operative proteins. © The Royal Society of Chemistry 2012.

  13. Calculation of nucleation free energy for duplex oligomers in the context of nearest neighbor models.

    Science.gov (United States)

    Guerra, João Carlos de Oliveira

    2013-08-01

    Additive physical properties of DNA double strand polymers have been expanded in terms of 8 irreducible parameters. This provided consistency relations among the corresponding 10 duplex dimer contributions. To allow for oligomer analysis, end parameters were often added, and this would add extra degrees of freedom to the fore mentioned parameters. Statistical mechanics approaches were then connected to the nearest neighbor (NN) approach in the framework of the two-states model. Ad hoc end effects were thus (wrongly) correlated to nucleation phenomena and this lead to a critique for its role in NN modeling. With this motivation, a new NN model is proposed that accommodates the nucleation free energies. The model relates the nucleation free energy to the mean composition of the chain and permits to obtain a good estimate for the free energy associated only to the Watson-Crick base pairings. Copyright © 2013 Wiley Periodicals, Inc.

  14. DNA nanostructures: Through, rather than across

    Science.gov (United States)

    Bruchez, Marcel P.

    2018-02-01

    Dye molecules are shown to assemble into J-aggregate arrays by sequence-specific organization in the minor groove of DNA duplex sequences. Energy transfer through these structures displays the hallmarks of coherent coupling over distances that exceed those of conventional dipole-coupling processes.

  15. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-08-03

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  16. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin–DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L.; Powers, Alvin C.; Liu, Guozheng

    2016-01-01

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ~95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  17. Duplex ultrasound: Indications and findings in a newly created ...

    African Journals Online (AJOL)

    2015-09-21

    Sep 21, 2015 ... Background: Peripheral limb vascular diseases form one of the most common diseases that affect the middle-aged and the ... author is credited and the new creations are licensed under the identical terms. .... Table 4: Relationship of major duplex ultrasound findings with age, gender and clinical information.

  18. ON SECURE FULL DUPLEX COMMUNICATION IN MOBILE AD HOC NETWORK

    OpenAIRE

    S. C. Dutta; Sudha Singh; D. K. Singh

    2013-01-01

    This paper is to establish Ad Hoc network in mobile phones and start fully secured full duplex communication in any situation. This type of communication will be cost effective and it will be fastest way of communication in case of any server failure or server error.

  19. Full-duplex wireless communications systems self-interference cancellation

    CERN Document Server

    Le-Ngoc, Tho

    2017-01-01

    This book introduces the development of self-interference (SI)-cancellation techniques for full-duplex wireless communication systems. The authors rely on estimation theory and signal processing to develop SI-cancellation algorithms by generating an estimate of the received SI and subtracting it from the received signal. The authors also cover two new SI-cancellation methods using the new concept of active signal injection (ASI) for full-duplex MIMO-OFDM systems. The ASI approach adds an appropriate cancelling signal to each transmitted signal such that the combined signals from transmit antennas attenuate the SI at the receive antennas. The authors illustrate that the SI-pre-cancelling signal does not affect the data-bearing signal. This book is for researchers and professionals working in wireless communications and engineers willing to understand the challenges of deploying full-duplex and practical solutions to implement a full-duplex system. Advanced-level students in electrical engineering and computer ...

  20. Risk Factors of Deep Venous Thrombosis in Duplex and Colour ...

    African Journals Online (AJOL)

    Risk Factors of Deep Venous Thrombosis in Duplex and Colour Doppler Ultrasound at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia. ... Thus early, correct and definitive diagnosis is crucial in assessing thromboembolic risk and initiating therapy. In this regard Patients at risk must be identified and given ...

  1. Full-Duplex Relay Selection in Cognitive Underlay Networks

    KAUST Repository

    Khafagy, Mohammad Galal

    2017-09-30

    In this work, we analyze the performance of full-duplex relay selection (FDRS) in spectrum-sharing networks. Contrary to half-duplex relaying, full-duplex relaying (FDR) enables simultaneous listening/forwarding at the secondary relay(s), thereby allowing for a higher spectral efficiency. However, since the source and relay simultaneously transmit in FDR, their superimposed signal at the primary receiver should now satisfy the existing interference constraint, which can considerably limit the secondary network throughput. In this regard, relay selection can offer an adequate solution to boost the secondary throughput while satisfying the imposed interference limit. We first analyze the performance of opportunistic FDRS with residual self-interference (RSI) by deriving the exact cumulative distribution function of its end-to-end signal-to-interference-plus-noise ratio under Nakagami-m fading. We also evaluate the offered diversity gain of relay selection for different full-duplex cooperation schemes in the presence/absence of a direct source-destination link. When the adopted RSI link gain model is sublinear in the relay power, which agrees with recent research findings, we show that remarkable diversity gain can be recovered even in the presence of an interfering direct link. Second, we evaluate the end-to-end performance of FDRS with interference constraints due to the presence of a primary receiver. Finally, the presented exact theoretical findings are verified by numerical simulations.

  2. Podiatry Ankle Duplex Scan: Readily Learned and Accurate in Diabetes.

    Science.gov (United States)

    Normahani, Pasha; Powezka, Katarzyna; Aslam, Mohammed; Standfield, Nigel J; Jaffer, Usman

    2018-03-01

    We aimed to train podiatrists to perform a focused duplex ultrasound scan (DUS) of the tibial vessels at the ankle in diabetic patients; podiatry ankle (PodAnk) duplex scan. Thirteen podiatrists underwent an intensive 3-hour long simulation training session. Participants were then assessed performing bilateral PodAnk duplex scans of 3 diabetic patients with peripheral arterial disease. Participants were assessed using the duplex ultrasound objective structured assessment of technical skills (DUOSATS) tool and an "Imaging Score". A total of 156 vessel assessments were performed. All patients had abnormal waveforms with a loss of triphasic flow. Loss of triphasic flow was accurately detected in 145 (92.9%) vessels; the correct waveform was identified in 139 (89.1%) cases. Participants achieved excellent DUOSATS scores (median 24 [interquartile range: 23-25], max attainable score of 26) as well as "Imaging Scores" (8 [8-8], max attainable score of 8) indicating proficiency in technical skills. The mean time taken for each bilateral ankle assessment was 20.4 minutes (standard deviation ±6.7). We have demonstrated that a focused DUS for the purpose of vascular assessment of the diabetic foot is readily learned using intensive simulation training.

  3. ON SECURE FULL DUPLEX COMMUNICATION IN MOBILE AD HOC NETWORK

    Directory of Open Access Journals (Sweden)

    S. C. Dutta

    2013-06-01

    Full Text Available This paper is to establish Ad Hoc network in mobile phones and start fully secured full duplex communication in any situation. This type of communication will be cost effective and it will be fastest way of communication in case of any server failure or server error.

  4. The down regulation of target genes by photo activated DNA nanoscissors.

    Science.gov (United States)

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  5. Development and validation of a duplex real-time PCR assay for the simultaneous detection of three mustard species (Sinapis alba, Brassica nigra and Brassica juncea) in food.

    Science.gov (United States)

    Palle-Reisch, Monika; Cichna-Markl, Margit; Hochegger, Rupert

    2014-06-15

    The paper presents a duplex real-time PCR assay for the simultaneous detection of three potentially allergenic mustard species commonly used in food: white mustard (Sinapis alba), black mustard (Brassica nigra) and brown mustard (Brassica juncea). White mustard is detected in the "green" and black/brown mustard in the "yellow" channel. The duplex real-time PCR assay does not show cross-reactivity with other Brassicaceae species including broccoli, cauliflower, radish and rapeseed. Low cross-reactivities (difference in the Ct value ⩾ 11.91 compared with the positive control) were obtained with cumin, fenugreek, ginger, rye and turmeric. When applying 500 ng DNA per PCR tube, the duplex real-time PCR assay allowed the detection of white, black and brown mustard in brewed model sausages down to a concentration of 5mg/kg in 10 out of 10 replicates. The duplex real-time PCR assay was applied to verify correct labelling of commercial foodstuffs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. On the Secrecy Degrees of Freedom with Full-Duplex Communication

    DEFF Research Database (Denmark)

    Mahmood, Nurul Huda; Mogensen, Preben Elgaard

    2017-01-01

    Full duplex communication enables simultaneous transmission from both ends of a communication link, thereby promising gains in terms of the throughput and the delay. Such compelling gains are conditioned on a number of design constraints. Generally, it has been shown that the throughput gain and ...... form expressions for the instantaneous and ergodic throughput gain of full duplex communication over conventional half duplex is derived. The secrecy degrees of freedom with full duplex is shown to be two as opposed to that of zero in half duplex mode....... of such an application. The potential of full duplex nodes in improving the physical layer security of a communication link is investigated in this contribution. We specifically derive the information theoretic secrecy degrees of freedom measure for a pair of nodes communicating in full duplex mode. Moreover, closed...

  7. Modulation of Interparticle Distance in Discrete Gold Nanoparticle Dimers and Trimers by DNA Single-Base Pairing.

    Science.gov (United States)

    Akiyama, Yoshitsugu; Shikagawa, Hiroto; Kanayama, Naoki; Takarada, Tohru; Maeda, Mizuo

    2015-07-01

    Self-assembled structures of metallic nanoparticles with dynamically changeable interparticle distance hold promise for the regulation of collective physical properties. This paper describes gold nanoparticle dimers and trimers that exhibit spontaneous and reversible changes in interparticle distance. To exploit this property, a gold nanoparticle is modified with precisely one long DNA strand and approximately five short DNA strands. The long DNA serves to align the nanoparticles on a template DNA via hybridization, while the short DNAs function to induce the interparticle distance changes. The obtained dimer and trimer are characterized with gel electrophoresis, dynamic light scattering measurements, and transmission electron microscopy (TEM). When the complementary short DNA is added to form the fully matched duplexes on the particle surface in the presence of MgCl2 , spontaneous reduction of the interparticle distance is observed with TEM and cryo-electron microscopy. By contrast, when the terminal-mismatched DNA is added, no structural change occurs under the same conditions. Therefore, the single base pairing/unpairing at the outermost surface of the nanoparticle impacts the interparticle distance. This unique feature could be applied to the regulation of structures and properties of various DNA-functionalized nanoparticle assemblies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway.

    Directory of Open Access Journals (Sweden)

    Peihong Dai

    2014-04-01

    Full Text Available Modified vaccinia virus Ankara (MVA is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs, which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs, but not in plasmacytoid dendritic cells (pDCs. Transcription factors IRF3 (IFN regulatory factor 3 and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1, are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase. MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1 and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

  9. Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

    Science.gov (United States)

    Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

    2017-09-15

    Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses. Copyright © 2017 American Society for Microbiology.

  10. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-14

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  11. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  12. The XRCC 1 DNA repair gene modifies the environmental risk of stomach cancer: a hospital-based matched case-control study.

    Science.gov (United States)

    Putthanachote, Nuntiput; Promthet, Supannee; Hurst, Cameron; Suwanrungruang, Krittika; Chopjitt, Peechanika; Wiangnon, Surapon; Chen, Sam Li-Sheng; Yen, Amy Ming-Fang; Chen, Tony Hsiu-Hsi

    2017-10-11

    Previous studies have found that polymorphisms of the DNA repair gene X-ray repair cross-complementing group 1(XRCC1) and environmental factors are both associated with an increased risk of stomach cancer, but no study has reported on the potential additive effect of these factors among Thai people. The aim of this study was to investigate whether the risk of stomach cancer from XRCC1 gene polymorphisms was modified by environmental factors in the Thai population. Hospital-based matched case-control study data were collected from 101 new stomach cancer cases and 202 controls, which were recruited from2002 to 2006 and were matched for gender and age. Genotype analysis was performed using real-time PCR-HRM. The data were analysed by the chi-square test and conditional logistic regression. The Arg/Arg homozygote polymorphism of the XRCC1 gene was associated with an increased risk of stomach cancer in the Thai population (OR adj , 3.7; 95%CI, 1.30-10.72) compared with Gln/Gln homozygosity. The effect of the XRCC1gene on the risk of stomach cancer was modified by both a high intake of vegetable oils and salt (p = 0.036 and p = 0.014), particularly for the Arg/Arg homozygous genotype. There were, however, no additive effects on the risk of stomach cancer between variants of the XRCC1gene and smoking,alcohol or pork oil consumption. The effect of the XRCC1 gene homozygosity, particularly Arg/Arg, on the risk for stomach cancer was elevated by a high intake of vegetable oils and salt.

  13. Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0.

    Science.gov (United States)

    Salpini, Romina; Piermatteo, Lorenzo; Gill, Upkar; Battisti, Arianna; Stazi, Francesca; Guenci, Tania; Giannella, Sara; Serafini, Valentina; Kennedy, Patrick T F; Perno, Carlo Federico; Svicher, Valentina; Ciotti, Marco

    2017-08-01

    Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R 2  = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R 2  = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

  14. Full-Duplex Communications in Large-Scale Cellular Networks

    KAUST Repository

    AlAmmouri, Ahmad

    2016-04-01

    In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization and efficiency. However, the penetration of FD communications to the cellular networks domain is challenging due to the imposed uplink/downlink interference. This thesis presents a tractable framework, based on stochastic geometry, to study FD communications in multi-tier cellular networks. Particularly, we assess the FD communications effect on the network performance and quantify the associated gains. The study proves the vulnerability of the uplink to the downlink interference and shows that the improved FD rate gains harvested in the downlink (up to 97%) comes at the expense of a significant degradation in the uplink rate (up to 94%). Therefore, we propose a novel fine-grained duplexing scheme, denoted as α-duplex scheme, which allows a partial overlap between the uplink and the downlink frequency bands. We derive the required conditions to harvest rate gains from the α-duplex scheme and show its superiority to both the FD and half-duplex (HD) schemes. In particular, we show that the α-duplex scheme provides a simultaneous improvement of 28% for the downlink rate and 56% for the uplink rate. We also show that the amount of the overlap can be optimized based on the network design objective. Moreover, backward compatibility is an essential ingredient for the success of new technologies. In the context of in-band FD communication, FD base stations (BSs) should support HD users\\' equipment (UEs) without sacrificing the foreseen FD gains. The results show that FD-UEs are not necessarily required to harvest rate gains from FD-BSs. In particular, the results show that adding FD-UEs to FD-BSs offers a maximum of 5% rate gain over FD-BSs and HD-UEs case, which is a marginal gain compared to the burden required to implement FD transceivers at the UEs\\' side. To this end, we shed light on practical scenarios where HD-UEs operation with FD-BSs outperforms the

  15. Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.

    Science.gov (United States)

    Yarranton, G T; Gefter, M L

    1979-04-01

    Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.

  16. Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification.

    Science.gov (United States)

    Xia, Ning; Liu, Ke; Zhou, Yingying; Li, Yuanyuan; Yi, Xinyao

    2017-01-01

    miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted "Fc-DNA-Fc") presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0-25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors.

  17. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  18. Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

    Science.gov (United States)

    Sun, Yan; Borbat, Peter P; Grigoryants, Vladimir M; Myers, William K; Freed, Jack H; Scholes, Charles P

    2015-02-17

    Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26-27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ~35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. In situ detection of tandem DNA repeat length

    Energy Technology Data Exchange (ETDEWEB)

    Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

    1996-11-01

    A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

  20. Tensile properties of duplex UNS S32205 and lean duplex UNS S32304 steels and the influence of short duration 475 ºC aging

    Directory of Open Access Journals (Sweden)

    Sérgio Souto Maior Tavares

    2012-12-01

    Full Text Available Duplex stainless steels are high strength and corrosion resistant steels extensively used in the petrochemical and chemical industries. The aging at 475 ºC for long periods of time provokes embrittlement and deterioration of corrosion resistance. However, short duration aging at 475 ºC may be used as heat treatment to improve mechanical resistance with small decrease in the other properties. In this work the flow stress curves of lean duplex UNS S32304 and duplex UNS S32205 steels were modeled with Hollomon's equation and work hardening exponents (n were determined. The analyses were conducted in specimens annealed and heat treated at 475 ºC for short periods of time. The aging at 475 ºC for 4 hours, 8 hours and 12 hours promoted significant hardening with small decrease of ductility. The work hardening exponents of both steels were compared, being higher in the duplex steel than in the lean duplex grade.

  1. Duplex-tube sodium-indication steam generator

    International Nuclear Information System (INIS)

    Matal, O.; Martoch, J.

    1984-01-01

    The steam generator with duplex tubes and sodium indication is connected to the main sodium input and output via the inlet and outlet chambers and has indication spaces connected to the interspaces of the duplex tubes. The first indication space is linked with the auxiliary inlet pipe to the inlet chamber and the second indication space is connected with the auxiliary pipe to the outlet chamber. Mounted to the auxiliary inlet pipe is at least one closure, i.e., a valve or electromagnetic stop. Mounted on the auxiliary outlet pipe is an indication sensor, e.g., a sodium flow sensor. At least one indication space is provided with an alarm sensor, e.g., a thermocouple, a pressure gauge and one sensor to monitor the hydrogen content of sodium. (J.P.)

  2. Differentiation of midbrain cystic lesions by duplex Doppler US

    International Nuclear Information System (INIS)

    Horgan, J.G.; Taylor, K.J.W.; Sarwar, M.; Weltin, G.; Klier, L.

    1986-01-01

    The addition of Doppler technology to an ultrasound imaging system allows easy differentiation of vascular from nonvascular masses. In addition, the characteristic flow in different arteries, veins, and arteriovenous communications allows accurate diagnosis of vascular structures. Cystic lesions in the region of the midbrain are uncommon but not rare in neonates, and it is important to differentiate between quadrigeminal plate cysts and vein of Galen aneurysms. Six neonates with cystic midbrain structures lying above the tectum were studied over a 2-year period. All were examined with duplex Doppler imaging and followed up by CT and angiography, when indicated. In two of the six infants a highly vascular signal was demonstrated with high flow rates and low impedance. Subsequent CT and angiography revealed typical vein of Galen aneurysms. The remaining four infants had cystic lesions that were avascular on Doppler imaging and demonstrated no enhancement on CT. Duplex Doppler imaging is helpful in the initial investigation of patients with midbrain cystic lesions

  3. Herpes Zoster Duplex Unilateralis: Two Cases and Brief Literature Review.

    Science.gov (United States)

    Son, Jee Hee; Chung, Bo Young; Kim, Hye One; Cho, Hee Jin; Park, Chun Wook

    2016-12-01

    Cases involving dermatomal herpes zoster in two or more locations are rare, especially in immunocompetent patients. When two noncontiguous dermatomes are involved, if affected unilaterally, it is called herpes zoster duplex unilateralis; if bilaterally, bilateralis. Here, we report two cases of herpes zoster duplex unilateralis. A 66-year-old man presented with painful erythematous grouped vesicles on his left scalp, forehead, trunk, and back (left [Lt.] V1, Lt. T8). Histologic findings were consistent with herpetic infection. A 33-year-old woman presented with painful erythematous grouped vesicles and crust on her left forehead and neck (Lt. V1, Lt. C5). Both patients were treated with oral administration of famcyclovir 750 mg/day for seven days.

  4. Listen and talk full-duplex cognitive radio networks

    CERN Document Server

    Liao, Yun; Han, Zhu

    2016-01-01

    This brief focuses on the use of full-duplex radio in cognitive radio networks, presenting a novel spectrum sharing protocol that allows the secondary users to simultaneously sense and access the vacant spectrum. This protocol, called “Listen-and-talk” (LAT), is evaluated by both mathematical analysis and computer simulations in comparison with other existing protocols, including the listen-before-talk protocol. In addition to LAT-based signal processing and resource allocation, the brief discusses techniques such as spectrum sensing and dynamic spectrum access. The brief proposes LAT as a suitable access scheme for cognitive radio networks, which can support the quality-of-service requirements of these high priority applications. Fundamental theories and key techniques of cognitive radio networks are also covered. Listen and Talk: Full-duplex Cognitive Radio Networks is designed for researchers, developers, and professionals involved in cognitive radio networks. Advanced-level students studying signal pr...

  5. A 2-order MIMO Full-Duplex Antenna System

    DEFF Research Database (Denmark)

    Tsakalaki, Elpiniki; Foroozanfard, Ehsan; De Carvalho, Elisabeth

    2014-01-01

    The paper presents an antenna system with combined full-duplex and 2-order multiple-input-multiple-output (MIMO) functionalities, i.e., a system capable of spatially multiplexing and spatially demultiplexing 2 datastreams in the same frequency and in the same time. By exploiting symmetries....... On the other hand, the 2 MIMO ports (either at the Tx or at the Rx) are sufficiently decoupled thanks to polarization diversity. The proposed antenna system exhibits a remarkable level of fullduplex isolation over a wide bandwidth while maintaining low coupling between its MIMO ports and can serve...... as a concrete implementation of an antenna system equipped with both MIMO as well as full-duplex capabilities....

  6. DNA-DNA interaction beyond the ground state.

    Science.gov (United States)

    Lee, D J; Wynveen, A; Kornyshev, A A

    2004-11-01

    The electrostatic interaction potential between DNA duplexes in solution is a basis for the statistical mechanics of columnar DNA assemblies. It may also play an important role in recombination of homologous genes. We develop a theory of this interaction that includes thermal torsional fluctuations of DNA using field-theoretical methods and Monte Carlo simulations. The theory extends and rationalizes the earlier suggested variational approach which was developed in the context of a ground state theory of interaction of nonhomologous duplexes. It shows that the heuristic variational theory is equivalent to the Hartree self-consistent field approximation. By comparison of the Hartree approximation with an exact solution based on the QM analogy of path integrals, as well as Monte Carlo simulations, we show that this easily analytically-tractable approximation works very well in most cases. Thermal fluctuations do not remove the ability of DNA molecules to attract each other at favorable azimuthal conformations, neither do they wash out the possibility of electrostatic "snap-shot" recognition of homologous sequences, considered earlier on the basis of ground state calculations. At short distances DNA molecules undergo a "torsional alignment transition," which is first order for nonhomologous DNA and weaker order for homologous sequences.

  7. Investigation of hot cracking resistance of 2205 duplex steel

    International Nuclear Information System (INIS)

    Adamiec, J; Scibisz, B

    2010-01-01

    Austenitic duplex steel of the brand 2205 according to Avesta Sheffield is used for welded constructions (pipelines, tanks) in the petrol industry, chemical industry and food industry. It is important to know the range of high-temperature brittleness in designing welding technology for constructions made of this steel type. There is no data in literature concerning this issue. High-temperature brittleness tests using the simulator of heat flow device Gleeble 3800 were performed. The tests results allowed the evaluation of the characteristic temperatures in the brittleness temperature range during the joining of duplex steels, specifically the nil-strength temperature (NST) and nil-ductility temperatures (NDT) during heating, the strength and ductility recovery temperatures (DRT) during cooling, the R f parameter (R f = (T liquidus - NDT)/NDT) describing the duplex steel inclination for hot cracking, and the brittleness temperature range (BTR). It has been stated that, for the examined steel, this range is wide and amounts to ca. 90 deg. C. The joining of duplex steels with the help of welding techniques creates a significant risk of hot cracks. After analysis of the DTA curves a liquidus temperature of T L = 1465 deg. C and a solidus temperature of T S = 1454 deg. C were observed. For NST a mean value was assumed, in which the cracks appeared for six samples; the temperature was 1381 deg. C. As the value of the NDT temperature 1367 deg. C was applied while for DRT the assumed temperature was 1375 deg. C. The microstructure of the fractures was observed using a Hitachi S-3400N scanning electron microscope (SEM). The analyses of the chemical composition were performed using an energy-dispersive X-ray spectrometer (EDS), Noran System Six of Thermo Fisher Scientific. Essential differences of fracture morphology type over the brittle temperature range were observed and described.

  8. Implementation of real-time duplex synthetic aperture ultrasonography

    DEFF Research Database (Denmark)

    Hemmsen, Martin Christian; Larsen, Lee; Kjeldsen, Thomas

    2015-01-01

    This paper presents a real-time duplex synthetic aperture imaging system, implemented on a commercially available tablet. This includes real-time wireless reception of ultrasound signals and GPU processing for B-mode and Color Flow Imaging (CFM). The objective of the work is to investigate the im...... and that the required bandwidth between the probe and processing unit is within the current Wi-Fi standards....

  9. Effect of heat treatment on corrosion behavior of duplex coatings

    Directory of Open Access Journals (Sweden)

    K. Raghu Ram Mohan Reddy

    2017-01-01

    Full Text Available In the present paper, duplex WC-Co/NiCrAlY coating is coated onto Ti6Al4V substrate and vacuum heat treatment is employed to investigate the corrosion behavior of heat treated samples as well as Ti6Al4V substrate for comparison. In this duplex coating system, High Velocity Oxy Fuel (HVOF process is used to deposit NiCrAlY interlayer with a constant thickness of 200 μm and WC-Co ceramic top layer with varying thickness of 250 μm, 350 μm and 450 μm deposited by Detonation Spray (DS process. Different heat treatment temperatures (600–1150 °C were employed for the coated samples to study the microstructure and the effect on corrosion resistance of the duplex coatings. Potentiodynamic polarization tests were carried to investigate the corrosion performance of duplex coated heat treated samples and the substrate in Ringer’s solution at 37 °C and prepared the pH to 5.7. The microstructure upon corrosion after heat treatment was characterized by SEM analysis to understand the corrosion behavior. The results disclosed that at all heat treatment temperatures, all the coated samples exhibited better corrosion resistance than the base substrate. However, during 950 °C and 1150 °C heat treatment temperatures, it was observed highest corrosion potential than 600 °C and 800 °C. The 350 μm thickness, coated sample exhibited highest corrosion resistance compared to other two coated samples and the substrate at all heat treatment temperatures.

  10. Herpes zoster duplex bilateralis in an immunocompetent host

    Directory of Open Access Journals (Sweden)

    Pratik Gahalaut

    2012-01-01

    Full Text Available Varicella zoster virus causes both chicken pox and herpes zoster. The phenomenon of herpes zoster occurring concurrently in two non-contiguous dermatomes involving different halves of the body is termed herpes zoster duplex bilateralis (HZDB. Few cases, reported in the literature, were seen in either an immunosuppressed host or in the older age group. Here we present a case of HZDB in an immunocompetent host, probably the first in India.

  11. Two-step mechanism for modifier of transcription 1 (Mot1) enzyme-catalyzed displacement of TATA-binding protein (TBP) from DNA.

    Science.gov (United States)

    Moyle-Heyrman, Georgette; Viswanathan, Ramya; Widom, Jonathan; Auble, David T

    2012-03-16

    The TATA box binding protein (TBP) is a central component of the transcription preinitiation complex, and its occupancy at a promoter is correlated with transcription levels. The TBP-promoter DNA complex contains sharply bent DNA and its interaction lifetime is limited by the ATP-dependent TBP displacement activity of the Snf2/Swi2 ATPase Mot1. Several mechanisms for Mot1 action have been proposed, but how it catalyzes TBP removal from DNA is unknown. To better understand the Mot1 mechanism, native gel electrophoresis and FRET were used to determine how Mot1 affects the trajectory of DNA in the TBP-DNA complex. Strikingly, in the absence of ATP, Mot1 acts to unbend DNA, whereas TBP remains closely associated with the DNA in a stable Mot1-TBP-DNA ternary complex. Interestingly, and in contrast to full-length Mot1, the isolated Mot1 ATPase domain binds DNA, and its affinity for DNA is nucleotide-dependent, suggesting parallels between the Mot1 mechanism and DNA translocation-based mechanisms of chromatin remodeling enzymes. Based on these findings, a model is presented for Mot1 that links a DNA conformational change with ATP-induced DNA translocation.

  12. Crystal structure of a four-stranded intercalated DNA: d(C4)

    Science.gov (United States)

    Chen, L.; Cai, L.; Zhang, X.; Rich, A.

    1994-01-01

    The crystal structure of d(C4) solved at 2.3-A resolution reveals a four-stranded molecule composed of two interdigitated or intercalated duplexes. The duplexes are held together by hemiprotonated cytosine-cytosine base pairs and are parallel stranded, but the two duplexes point in opposite directions. The molecule has a slow right-handed twist of 12.4 degrees between covalently linked cytosine base pairs, and the base stacking distance is 3.1 A. This is in general agreement with the NMR studies. A biological role for DNA in this conformation is suggested.

  13. Development and test evaluation of duplex steam reformer tube

    International Nuclear Information System (INIS)

    Allen, D.C.; Meyer, D.J.; Pflasterer, G.R.

    1980-01-01

    For HTR applications involving a steam reformer (SR), it is uncertain whether an intermediate heat exchanger (IHX) is required. There are several system configurations that could be developed for the application of nuclear heat using the steam reformer reaction. The considerations (advantages vs. disadvantages) for each of the system configurations are summarized. The approach that technically and economically appears to be the most attractive, in studies conducted by General Electric, combines the SR process heat exchanger and the IHX in a single component using a duplex tube. A central question concerning the duplex tube concept is whether the design would provide adequate leak monitoring capability and significant reduction in tritium and hydrogen diffusion, while introducing only a small increase in overall temperature difference from the helium to the process gas. A cooperative GE-KFA effort was undertaken to develop, fabricate, test, and evaluate a duplex steam reformer tube. GE was responsible for the development and fabrication of the tube, and KFA was responsible for testing the tube in the EVA I facility at Juelich. Both GE and KFA are evaluating the thermochemical and metallurgical test data. Actual fabrication of the tube was performed by Foster-Wheeler in accordance with the GE design. This paper reviews the highlights of the fabrication development and preliminary evaluation of the test data

  14. On full duplex Gaussian relay channels with self-interference

    KAUST Repository

    Behboodi, Arash

    2016-08-15

    Self interference (SI) in full duplex (FD) systems is the interference caused by the transmission stream on the reception stream. Being one of the main restrictive factors for performance of practical full duplex systems, however, not too much is known about its effect on the fundamental limits of relaying systems. In this work, we consider the full duplex three-node relay channel with SI where SI is modeled as an additive Gaussian noise whose variance is dependent on instantaneous input power. The classical achievable rates and upper bounds for the single three-node relay channel no longer apply due to the structure of SI. Achievable rates for Decode-and-Forward (DF) and Compress-and-Forward (CF) and upper bounds on the capacity are derived assuming Gaussian inputs and SI. The deterministic model is also introduced and its capacity is characterized. The optimal joint source-relay distributions is discussed. Numerical results are provided comparing the achievable rates and upper bound. © 2016 IEEE.

  15. Duplex geometry: an example from the Moine Thrust Belt

    Science.gov (United States)

    Bowler, S.

    1987-04-01

    The geometry and microstructure of a small duplex formed in one bed from the Moine Thrust Belt of northwest Scotland is reported. The structure is seen in oblique section, within the Cambrian Pipe-rock, in an area of low strain. A range of movement direction indicators are present in the structure. An early grain shape fabric developed close to the roof thrust is taken as the best estimate of the overall movement direction towards 287°. Slickensides in the gouge developed on movement planes within the duplex show varied orientations on a given plane, and are not considered useful indicators of thrust transport direction. Branch lines exposed converge and diverge, suggesting little lateral continuity of the exposed structure. The microstructures present within the structure indicate an increase in localised deformation, and in cataclastic behavior as the duplex evolved. Early layer parallel shear is ubiquitous, giving rise to an elongate grain shape fabric close to bedding surfaces. In early formed horses, a layer-parallel, oblate grain shape fabric, which shows localised slip zones, is overprinted by gouge formation. Later formed horses show only fracturing and gouge development. This sequence is attributed to stick-slip behavior in the propagation or displacement of the original fault, now the floor thrust.

  16. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  17. Enzymatic recognition of DNA replication origins

    International Nuclear Information System (INIS)

    Stayton, M.M.; Bertsch, L.; Biswas, S.

    1983-01-01

    In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

  18. EFFECT OF INTERMETALLIC PHASES ON CORROSION BEHAVIOR AND MECHANICAL PROPERTIES OF DUPLEX STAINLESS STEEL AND SUPER-DUPLEX STAINLESS STEEL

    Directory of Open Access Journals (Sweden)

    Prabhu Paulraj

    2015-08-01

    Full Text Available Duplex Stainless Steels (DSS and Super Duplex Stainless Steel (SDSS have excellent integration of mechanical and corrosion properties. However, the formation of intermetallic phases is a major problem in their usage. The mechanical and corrosion properties are deteriorated due to the presence of intermetallic phases. These phases are induced during welding, prolonged exposure to high temperatures, and improper heat treatments. The main emphasis of this review article is on intermetallic phases and their effects on corrosion and mechanical properties. First the effect of various alloying elements on DSS and SDSS has been discussed followed by formation of various intermetallic phases. The intermetallic phases affect impact toughness and corrosion resistance significantly. Their deleterious effect on weldments has also been reviewed.

  19. Characterization of microstructure and texture across dissimilar super duplex/austenitic stainless steel weldment joint by super duplex filler metal

    Energy Technology Data Exchange (ETDEWEB)

    Eghlimi, Abbas, E-mail: a.eghlimi@ma.iut.ac.ir [Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Shamanian, Morteza [Department of Materials Engineering, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Eskandarian, Masoomeh [Department of Materials Engineering, Shiraz University, Shiraz 71348-51154 (Iran, Islamic Republic of); Zabolian, Azam [Department of Natural Resources, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Szpunar, Jerzy A. [Department of Mechanical Engineering, University of Saskatchewan, Saskatoon SK S7N 5A9 (Canada)

    2015-08-15

    In the present paper, microstructural changes across an as-welded dissimilar austenitic/duplex stainless steel couple welded by a super duplex stainless steel filler metal using gas tungsten arc welding process is characterized with optical microscopy and electron back-scattered diffraction techniques. Accordingly, variations of microstructure, texture, and grain boundary character distribution of base metals, heat affected zones, and weld metal were investigated. The results showed that the weld metal, which was composed of Widmanstätten austenite side-plates and allotriomorphic grain boundary austenite morphologies, had the weakest texture and was dominated by low angle boundaries. The welding process increased the ferrite content but decreased the texture intensity at the heat affected zone of the super duplex stainless steel base metal. In addition, through partial ferritization, it changed the morphology of elongated grains of the rolled microstructure to twinned partially transformed austenite plateaus scattered between ferrite textured colonies. However, the texture of the austenitic stainless steel heat affected zone was strengthened via encouraging recrystallization and formation of annealing twins. At both interfaces, an increase in the special character coincident site lattice boundaries of the primary phase as well as a strong texture with <100> orientation, mainly of Goss component, was observed. - Graphical abstract: Display Omitted - Highlights: • Weld metal showed local orientation at microscale but random texture at macroscale. • Intensification of <100> orientated grains was observed adjacent to the fusion lines. • The austenite texture was weaker than that of the ferrite in all duplex regions. • Welding caused twinned partially transformed austenites to form at SDSS HAZ. • At both interfaces, the ratio of special CSL boundaries of the primary phase increased.

  20. Residual stresses and fatigue in a duplex stainless steel

    International Nuclear Information System (INIS)

    Johansson, Johan

    1999-01-01

    Duplex stainless steels, consisting of approximately equal amounts of austenite and ferrite, often combine the best features of austenitic and ferritic stainless steels. They generally have good mechanical properties, including high strength and ductility, and the corrosion resistance is often better than conventional austenitic grades. This has lead to a growing use of duplex stainless steels as a material in mechanically loaded constructions. However, detailed knowledge regarding its mechanical properties and deformation mechanisms are still lacking. In this thesis special emphasis has been placed on the residual stresses and their influence on mechanical behaviour of duplex stainless steels. Due to the difference in coefficient of thermal expansion between the two phases, tensile microstresses are found in the austenitic phase and balancing compressive microstresses in the ferritic phase. The first part of this thesis is a literature survey, which will give an introduction to duplex stainless steels and review the fatigue properties of duplex stainless steels and the influence of residual stresses in two-phase material. The second part concerns the evolution of the residual stress state during uniaxial loading. Initial residual stresses were found to be almost two times higher in the transverse direction compared to the rolling direction. During loading the absolute value of the microstresses increased in the macroscopic elastic regime but started to decrease with increasing load in the macroscopic plastic regime. A significant increase of the microstresses was also found to occur during unloading. Finite element simulations also show stress variation within one phase and a strong influence of both the elastic and plastic anisotropy of the individual phases on the simulated stress state. In the third part, the load sharing between the phases during cyclic loading is studied. X-ray diffraction stress analysis and transmission electron microscopy show that even if

  1. Residual stresses and fatigue in a duplex stainless steel

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, Johan

    1999-05-01

    Duplex stainless steels, consisting of approximately equal amounts of austenite and ferrite, often combine the best features of austenitic and ferritic stainless steels. They generally have good mechanical properties, including high strength and ductility, and the corrosion resistance is often better than conventional austenitic grades. This has lead to a growing use of duplex stainless steels as a material in mechanically loaded constructions. However, detailed knowledge regarding its mechanical properties and deformation mechanisms are still lacking. In this thesis special emphasis has been placed on the residual stresses and their influence on mechanical behaviour of duplex stainless steels. Due to the difference in coefficient of thermal expansion between the two phases, tensile microstresses are found in the austenitic phase and balancing compressive microstresses in the ferritic phase. The first part of this thesis is a literature survey, which will give an introduction to duplex stainless steels and review the fatigue properties of duplex stainless steels and the influence of residual stresses in two-phase material. The second part concerns the evolution of the residual stress state during uniaxial loading. Initial residual stresses were found to be almost two times higher in the transverse direction compared to the rolling direction. During loading the absolute value of the microstresses increased in the macroscopic elastic regime but started to decrease with increasing load in the macroscopic plastic regime. A significant increase of the microstresses was also found to occur during unloading. Finite element simulations also show stress variation within one phase and a strong influence of both the elastic and plastic anisotropy of the individual phases on the simulated stress state. In the third part, the load sharing between the phases during cyclic loading is studied. X-ray diffraction stress analysis and transmission electron microscopy show that even if

  2. Extended Life Testing of Duplex Ball Bearings

    Science.gov (United States)

    Mobley, Jeffrey; Robertson, Michael; Hodges, Charles

    2016-01-01

    Sierra Nevada Corporation’s Space Systems performed bearing life testing for the Scan Mirror Motor/Encoder Assembly (SMMA), part of the Scan Mirror Assembly on-board the Aerosol Polarimetry Sensor (APS) on the NASA Glory Spacecraft. The baseline bearing life test duration extended beyond the launch date for the Glory Spacecraft; a risk that the program was willing to undertake with the understanding that if any anomalies or failures occurred before the required life was achieved, then the mission objectives or operating profile could be modified on orbit to take those results into account. Even though the Glory Spacecraft failed to reach orbit during its launch in March of 2011, the bearing life testing was continued through a mutual understanding of value between Sierra Nevada Corporation and our customer; with a revised goal of testing to failure rather than completing a required number of life cycles. Life testing thus far has not only exceeded the original mission required life, but has also exceeded the published test data for Cumulative Degradation Factor (CDF) from NASA/CR-2009-215681. Many lessons were learned along the way regarding long life testing. The bearing life test has been temporarily suspended due to test support equipment issues.

  3. Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III.

    Science.gov (United States)

    Yakovleva, Lyudmila; Handy, Christopher J; Yagi, Haruhiko; Sayer, Jane M; Jerina, Donald M; Shuman, Stewart

    2006-06-20

    Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.

  4. Effect of intercalator substituent and nucleotide sequence on the stability of DNA- and RNA-naphthalimide complexes.

    Science.gov (United States)

    Johnson, Charles A; Hudson, Graham A; Hardebeck, Laura K E; Jolley, Elizabeth A; Ren, Yi; Lewis, Michael; Znosko, Brent M

    2015-07-01

    DNA intercalators are commonly used as anti-cancer and anti-tumor agents. As a result, it is imperative to understand how changes in intercalator structure affect binding affinity to DNA. Amonafide and mitonafide, two naphthalimide derivatives that are active against HeLa and KB cells in vitro, were previously shown to intercalate into DNA. Here, a systematic study was undertaken to change the 3-substituent on the aromatic intercalator 1,8-naphthalimide to determine how 11 different functional groups with a variety of physical and electronic properties affect binding of the naphthalimide to DNA and RNA duplexes of different sequence compositions and lengths. Wavelength scans, NMR titrations, and circular dichroism were used to investigate the binding mode of 1,8-naphthalimide derivatives to short synthetic DNA. Optical melting experiments were used to measure the change in melting temperature of the DNA and RNA duplexes due to intercalation, which ranged from 0 to 19.4°C. Thermal stabilities were affected by changing the substituent, and several patterns and idiosyncrasies were identified. By systematically varying the 3-substituent, the binding strength of the same derivative to various DNA and RNA duplexes was compared. The binding strength of different derivatives to the same DNA and RNA sequences was also compared. The results of these comparisons shed light on the complexities of site specificity and binding strength in DNA-intercalator complexes. For example, the consequences of adding a 5'-TpG-3' or 5'-GpT-3' step to a duplex is dependent on the sequence composition of the duplex. When added to a poly-AT duplex, naphthalimide binding was enhanced by 5.6-11.5°C, but when added to a poly-GC duplex, naphthalimide binding was diminished by 3.2-6.9°C. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

    Science.gov (United States)

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-01-01

    DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

  6. Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

    Directory of Open Access Journals (Sweden)

    Yasuaki Enokida

    Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

  7. Duplex ultrasound scanning of peripheral arterial disease of the lower limb

    DEFF Research Database (Denmark)

    Eiberg, Jonas Peter; Rasmussen, John Bøje Grønvall; Hansen, Marc Allan

    2010-01-01

    To assess the reliability and applicability of duplex ultrasound scanning (DUS) of lower limb arteries, compared with digital subtraction angiography (DSA), in patients with peripheral arterial disease (PAD)....

  8. Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide.

    Science.gov (United States)

    Srikannathasan, Velupillai; Wohlkonig, Alexandre; Shillings, Anthony; Singh, Onkar; Chan, Pan F; Huang, Jianzhong; Gwynn, Michael N; Fosberry, Andrew P; Homes, Paul; Hibbs, Martin; Theobald, Andrew J; Spitzfaden, Claus; Bax, Benjamin D

    2015-10-01

    Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.

  9. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Directory of Open Access Journals (Sweden)

    Steven W Paugh

    2016-02-01

    Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  10. Molecular mechanisms of DNA photodamage

    Energy Technology Data Exchange (ETDEWEB)

    Starrs, S.M

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA){sub n} and (GA){sub n}, and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a

  11. Molecular mechanisms of DNA photodamage

    International Nuclear Information System (INIS)

    Starrs, S.M.

    2000-05-01

    Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA) n and (GA) n , and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a dimeric

  12. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Science.gov (United States)

    Moore, Elaine Ann; Xu, Yao-Zhong

    2017-01-01

    The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  13. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Directory of Open Access Journals (Sweden)

    Elaine Ann Moore

    Full Text Available The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  14. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    Science.gov (United States)

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms.

  15. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA...... structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic...

  16. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

    Science.gov (United States)

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; Saeed, Ayman; Abbas, Mohammad Nooredeen; El-Shahawi, Mohammad S.; Bashammakh, Abdulaziz S.; Alyoubi, Abdulrahman O.; O´Sullivan, Ciara K.

    2016-01-01

    Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10−11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need. PMID:27886248

  17. A fluorescent sensor based on methyldopa drug modified γ-Fe2O3 nanoparticles for ultrasensitive detection of calf thymus DNA

    Science.gov (United States)

    Shahabadi, Nahid; Maghsudi, Maryam; Kashanian, Soheila

    2016-03-01

    We reported the study of calf thymus DNA (ct-DNA) adsorption by the polymer of methyldopa (2-amino-3-(3,4-dihydroxyphenyl)-2-methyl acid, propanoic) (PMDP), magnetofluorescent PMDP-γ-Fe2O3 nanocrystal. The method is based on the extraordinarily high quenching efficiency of ct-DNA and the specific interaction between ct-DNA and PMDP-γ-Fe2O3 via guanine base and metal coordination, probably. It was found that the designed magnetic nanoparticles can adsorb ct-DNA in nM levels in the presence of NaCl and KCl. In acetate and phosphate buffers DNA were adsorbed completely. Also, we found that pH plays an important role in DNA adsorption onto PMDP-γ-Fe2O3 nanocrystal. PMDP-γ-Fe2O3 nanocrystal is highly hydrophilic and DNA desorption wasn't observed. We believe this study will further stimulate the application of PMDP-γ-Fe2O3 nanocrystal in bioanalytical chemistry and nanotechnology. PMDP-γ-Fe2O3 nanocrystal possesses the ability to interact with ct-DNA via a partial intercalative binding mechanism, as demonstrated by fluorescence displacement experiments and a significant red shift (ca, 10 nm) in UV-vis spectra.

  18. Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

    International Nuclear Information System (INIS)

    Swasey, Steven M; Gwinn, Elisabeth G

    2016-01-01

    The thermal and chemical fragility of DNA nanomaterials assembled by Watson–Crick (WC) pairing constrain the settings in which these materials can be used and how they can be functionalized. Here we investigate use of the silver cation, Ag + , as an agent for more robust, metal-mediated self-assembly, focusing on the simplest duplex building blocks that would be required for more elaborate Ag + –DNA nanostructures. Our studies of Ag + -induced assembly of non-complementary DNA oligomers employ strands of 2–24 bases, with varied base compositions, and use electrospray ionization mass spectrometry to determine product compositions. High yields of duplex products containing narrowly distributed numbers of Ag + can be achieved by optimizing solution conditions. These Ag + -mediated duplexes are stable to at least 60 mM Mg 2+ , higher than is necessary for WC nanotechnology schemes such as tile assemblies and DNA origami, indicating that sequential stages of Ag + -mediated and WC-mediated assembly may be feasible. Circular dichroism spectroscopy suggests simple helical structures for Ag + -mediated duplexes with lengths to at least 20 base pairs, and further indicates that the structure of cytosine-rich duplexes is preserved at high urea concentrations. We therefore propose an approach towards dynamic DNA nanomaterials with enhanced thermal and chemical stability through designs that combine sturdy silver-mediated ‘frames’ with WC paired ‘pictures’. (paper)

  19. Numerical modeling and optimization of machining duplex stainless steels

    Directory of Open Access Journals (Sweden)

    Rastee D. Koyee

    2015-01-01

    Full Text Available The shortcomings of the machining analytical and empirical models in combination with the industry demands have to be fulfilled. A three-dimensional finite element modeling (FEM introduces an attractive alternative to bridge the gap between pure empirical and fundamental scientific quantities, and fulfill the industry needs. However, the challenging aspects which hinder the successful adoption of FEM in the machining sector of manufacturing industry have to be solved first. One of the greatest challenges is the identification of the correct set of machining simulation input parameters. This study presents a new methodology to inversely calculate the input parameters when simulating the machining of standard duplex EN 1.4462 and super duplex EN 1.4410 stainless steels. JMatPro software is first used to model elastic–viscoplastic and physical work material behavior. In order to effectively obtain an optimum set of inversely identified friction coefficients, thermal contact conductance, Cockcroft–Latham critical damage value, percentage reduction in flow stress, and Taylor–Quinney coefficient, Taguchi-VIKOR coupled with Firefly Algorithm Neural Network System is applied. The optimization procedure effectively minimizes the overall differences between the experimentally measured performances such as cutting forces, tool nose temperature and chip thickness, and the numerically obtained ones at any specified cutting condition. The optimum set of input parameter is verified and used for the next step of 3D-FEM application. In the next stage of the study, design of experiments, numerical simulations, and fuzzy rule modeling approaches are employed to optimize types of chip breaker, insert shapes, process conditions, cutting parameters, and tool orientation angles based on many important performances. Through this study, not only a new methodology in defining the optimal set of controllable parameters for turning simulations is introduced, but also

  20. Weld failure analysis of 2205 duplex stainless steel nozzle

    Directory of Open Access Journals (Sweden)

    Jingqiang Yang

    2014-10-01

    Full Text Available Failure analyses of weld joint between the nozzle and the head of the reactor made of 2205 duplex stainless steel was performed by optical microscopy (OM and scanning electron microscopy (SEM. Cracks were found in HAZ of the weld. The depth of the cracks is equal to the thickness of the inner weld. Localized uneven distribution of ferrite/austenite with 80–90% ferrite in weld is found. Results show that the cracks occurred along columnar granular with cleavage fracture. Poor weld process probably results in these cracks.