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Sample records for dna direct chemical

  1. DNA adducts-chemical addons

    PubMed Central

    Rajalakshmi, T. R.; AravindhaBabu, N.; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  2. DNA adducts-chemical addons.

    PubMed

    Rajalakshmi, T R; AravindhaBabu, N; Shanmugam, K T; Masthan, K M K

    2015-04-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  3. Synthesis of chemically modified DNA.

    PubMed

    Shivalingam, Arun; Brown, Tom

    2016-06-15

    Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved. This review highlights methods currently used for the synthesis of moderately long chemically modified nucleic acids (up to 1000 bp), their limitations and areas for future expansion. PMID:27284032

  4. DNA sequencing: chemical methods

    SciTech Connect

    Ambrose, B.J.B.; Pless, R.C.

    1987-01-01

    Limited base-specific or base-selective cleavage of a defined DNA fragment yields polynucleotide products, the length of which correlates with the positions of the particular base (or bases) in the original fragment. Sverdlov and co-workers recognized the possibility of using this principle for the determination of DNA sequences. In 1977 a fully elaborated method was introduced based on this principle, which allowed routine analysis of DNA sequences over distances greater than 100 nucleotide unite from a defined, radiolabeled terminus. Six procedures for partial cleavage were described. Simultaneous parallel resolution of an appropriate set of partial cleavage mixtures by polyacrylamide gel electrophoresis, followed by visualization of the radioactive bands by autoradiography, allows the deduction of nucleotide sequence.

  5. Direct ionization of DNA in solution

    SciTech Connect

    Miller, J.H.; Wilson, W.E.; Ritchie, R.H.

    1993-07-01

    Most of the energy absorbed in the cell nucleus from a radiation field goes into the aqueous medium that surrounds macromolecules, like DNA, which are critical to the normal function of cells. This part of the energy deposition produces numerous reactive species that can diffuse to DNA sequences and induce chemical changes. The average diffusion distance of the free radicals that mediate this indirect mode of DNA damage is only a few nanometers because the cellular medium contains a high concentration of molecules that rapidly scavenge the radiation-induced species. Under these conditions, direct interaction of the radiation field with DNA can not be neglected as a potential mode of damage induction. Two aspects of the direct effect are discussed in this paper: (1) screening of the interaction between DNA and charged particles by the dielectric response of the aqueous medium and (2) the impact-parameter dependence of these interactions.

  6. Recognition and repair of chemically heterogeneous structures at DNA ends

    PubMed Central

    Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.; Tumbale, Percy; Williams, R. Scott

    2014-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  7. Recognition and repair of chemically heterogeneous structures at DNA ends.

    PubMed

    Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D; Tumbale, Percy; Williams, R Scott

    2015-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  8. Chemical approaches to DNA nanotechnology.

    PubMed

    Endo, Masayuki; Sugiyama, Hiroshi

    2009-10-12

    Due to its self-assembling nature, DNA is undoubtedly an excellent molecule for the creation of various multidimensional nanostructures and the placement of functional molecules and materials. DNA molecules behave according to the programs of their sequences. Mixtures of numbers of DNA molecules can be placed precisely and organized into single structures to form nanoarchitectures. Once the appropriate sequences for the target nanostructure are established, the predesigned structure can be built up by self-assembly of the designed DNA strands. DNA nanotechnology has already reached the stage at which the organization of desired functional molecules and nanomaterials can be programmed on a defined DNA scaffold. In this review, we will focus on DNA nanotechnology and describe the potential of synthetic chemistry to contribute to the further development of DNA nanomaterials. PMID:19714700

  9. RNA-directed DNA methylation in plants.

    PubMed

    Movahedi, Ali; Sun, Weibu; Zhang, Jiaxin; Wu, Xiaolong; Mousavi, Mohaddesseh; Mohammadi, Kourosh; Yin, Tongming; Zhuge, Qiang

    2015-11-01

    In plants, many small interfering RNAs (siRNAs) direct de novo methylation by DNA methyltransferase. DNA methylation typically occurs by RNA-directed DNA methylation (RdDM), which directs transcriptional gene silencing of transposons and endogenous transgenes. RdDM is driven by non-coding RNAs (ncRNAs) produced by DNA-dependent RNA polymerases IV and V (PolIV and PolV). The production of siRNAs is initiated by PolIV and ncRNAs produced by PolIV are precursors of 24-nucleotide siRNAs. In contrast, ncRNAs produced by PolV are involved in scaffolding RNAs. In this review, we summarize recent studies of RdDM. In particular, we focus on the mechanisms involved in chromatin remodeling by PolIV and PolV. PMID:26183954

  10. The structure of DNA by direct imaging

    PubMed Central

    Marini, Monica; Falqui, Andrea; Moretti, Manola; Limongi, Tania; Allione, Marco; Genovese, Alessandro; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Gentile, Francesco; Candeloro, Patrizio; Di Fabrizio, Enzo

    2015-01-01

    The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations. PMID:26601243

  11. The structure of DNA by direct imaging.

    PubMed

    Marini, Monica; Falqui, Andrea; Moretti, Manola; Limongi, Tania; Allione, Marco; Genovese, Alessandro; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Gentile, Francesco; Candeloro, Patrizio; Di Fabrizio, Enzo

    2015-08-01

    The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations. PMID:26601243

  12. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions. PMID:26014116

  13. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  14. T7 replisome directly overcomes DNA damage

    PubMed Central

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-01-01

    Cells and viruses possess several known ‘restart' pathways to overcome lesions during DNA replication. However, these ‘bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase–DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly. PMID:26675048

  15. T7 replisome directly overcomes DNA damage

    NASA Astrophysics Data System (ADS)

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-12-01

    Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

  16. Chemical Information: Print. Directed Study.

    ERIC Educational Resources Information Center

    Stone, Catherine C.

    This report provides a survey and evaluation of chemical information literature. Contained in this survey are an overview of the chemical literature field, comments on obtaining access to this literature and annotated bibliographies of primary, secondary, and tertiary sources as well as special topics. Primary sources include journals, patents,…

  17. Programmable chemical controllers made from DNA

    PubMed Central

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2014-01-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language', and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents. PMID:24077029

  18. Programmable chemical controllers made from DNA.

    PubMed

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2013-10-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents. PMID:24077029

  19. Programmable chemical controllers made from DNA

    NASA Astrophysics Data System (ADS)

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2013-10-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents.

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

    PubMed

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

    2013-01-01

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

  1. Robust 3D DNA FISH Using Directly Labeled Probes

    PubMed Central

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

    2013-01-01

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

  2. Determining orientation and direction of DNA sequences

    DOEpatents

    Goodwin, Edwin H.; Meyne, Julianne

    2000-01-01

    Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

  3. The fate of the chemical warfare agent during DNA extraction.

    PubMed

    Wilkinson, Della A; Hulst, Albert G; de Reuver, Leo P J; van Krimpen, Simon H; van Baar, Ben M L

    2007-11-01

    Forensic laboratories do not have the infrastructure to process or store contaminated DNA samples that have been recovered from a crime scene contaminated with chemical or biological warfare agents. Previous research has shown that DNA profiles can be recovered from blood exposed to several chemical warfare agents after the agent has been removed. The fate of four toxic agents, sulfur mustard, sodium 2-fluoroacetate, sarin, and diazinon, in a lysis buffer used in Promega DNA IQ extraction protocol was studied to determine if extraction would render the samples safe. Two independent analytical methods were used per agent, selected from GC-MS, 1H NMR, 19F NMR, (31)P NMR, or LC-ES MS. The methods were validated before use. Determinations were carried out in a semi-quantitative way, by direct comparison to standards. Agent levels in the elution buffer were found to be below the detectable limits for mustard, sarin, sodium 2-fluoroacetate or low (<0.02 mg/mL) for diazinon. Therefore, once extracted these DNA samples could be safely processed in a forensic laboratory. PMID:18093062

  4. Comparisons of direct extraction methods of microbial DNA from different paddy soils.

    PubMed

    Islam, Md Rashedul; Sultana, Tahera; Melvin Joe, M; Cho, Jang-Cheon; Sa, Tongmin

    2012-07-01

    Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18-20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical-enzymatic-mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A260/A230 and A260/A280 ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical-enzymatic and chemical-mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals. PMID:23961194

  5. Direct Detection and Sequencing of Damaged DNA Bases

    PubMed Central

    2011-01-01

    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

  6. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  7. Enhancing DNA Crystal Durability through Chemical Crosslinking.

    PubMed

    Zhang, Diana; Paukstelis, Paul J

    2016-06-16

    Three-dimensional (3D) DNA crystals have been envisioned as a powerful tool for the positional control of biological and non-biological arrays on the nanoscale. However, most DNA crystals contain short duplex regions that can result in low thermal stability. Additionally, because DNA is a polyanion, DNA crystals often require high cation concentrations to maintain their integrity. Here, we demonstrate that a DNA alkylating mustard, bis(2-chloroethyl)amine, can form interstrand crosslinks within a model 3D DNA crystal. The crosslinking procedure did not alter crystal X-ray diffraction properties, but it did significantly improve the overall stability of the crystals under a variety of conditions. Crosslinked crystals showed enhanced stability at elevated temperature and were stable at Mg(2+) concentrations as low as 1 mm. Remarkably, the crosslinked crystals showed significant resistance to DNase I treatment, while also having improved longevity in tissue culture mediums. Characterization of the crosslinked species suggest that there are multiple crosslinking sites, but that the most prevalent interstrand crosslink involves an unpaired 3'-terminal guanosine residue. The improved stability of these DNA crystals suggests that simple treatment with alkylating reagents might be sufficient to stabilize crystals and other DNA constructs for improved functionality in biological and non-biological applications. PMID:27108768

  8. Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

    PubMed

    Wu, Yiping; Zhang, Bintian; Guo, Liang-Hong

    2013-07-16

    Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes. PMID:23777269

  9. Chemical method for introducing haptens on to DNA probes

    SciTech Connect

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

    1988-05-01

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

  10. Directed Chemical Evolution with an Outsized Genetic Code

    PubMed Central

    Krusemark, Casey J.; Tilmans, Nicolas P.; Brown, Patrick O.; Harbury, Pehr B.

    2016-01-01

    The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution. PMID:27508294

  11. Directed Chemical Evolution with an Outsized Genetic Code.

    PubMed

    Krusemark, Casey J; Tilmans, Nicolas P; Brown, Patrick O; Harbury, Pehr B

    2016-01-01

    The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution. PMID:27508294

  12. Site-directed, on-surface assembly of DNA nanostructures.

    PubMed

    Meyer, Rebecca; Saccà, Barbara; Niemeyer, Christof M

    2015-10-01

    Two-dimensional DNA lattices have been assembled from DNA double-crossover (DX) motifs on DNA-encoded surfaces in a site-specific manner. The lattices contained two types of single-stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface-bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA-tethered probe molecules on the opposite side of the lattices exposed to the solution. Site-specific lattice assembly and attachment of fluorophore-labeled oligonucleotides and DNA-protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications. PMID:26306556

  13. DNA Charge Transport: from Chemical Principles to the Cell.

    PubMed

    Arnold, Anna R; Grodick, Michael A; Barton, Jacqueline K

    2016-01-21

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

  14. Directly labeled fluorescent DNA probes for chromosome mapping

    SciTech Connect

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

    1995-12-31

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

  15. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  16. Direct surface-enhanced Raman scattering analysis of DNA duplexes.

    PubMed

    Guerrini, Luca; Krpetić, Željka; van Lierop, Danny; Alvarez-Puebla, Ramon A; Graham, Duncan

    2015-01-19

    The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface-enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label-free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5-methylated cytosine and N6-methylated Adenine) in duplexes. PMID:25414148

  17. Novel selection methods for DNA-encoded chemical libraries

    PubMed Central

    Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

    2015-01-01

    Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    PubMed

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

    2016-02-19

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

  20. Mesoporous silica nanoparticles deliver DNA and chemicals into plants

    NASA Astrophysics Data System (ADS)

    Torney, François; Trewyn, Brian G.; Lin, Victor S.-Y.; Wang, Kan

    2007-05-01

    Surface-functionalized silica nanoparticles can deliver DNA and drugs into animal cells and tissues. However, their use in plants is limited by the cell wall present in plant cells. Here we show a honeycomb mesoporous silica nanoparticle (MSN) system with 3-nm pores that can transport DNA and chemicals into isolated plant cells and intact leaves. We loaded the MSN with the gene and its chemical inducer and capped the ends with gold nanoparticles to keep the molecules from leaching out. Uncapping the gold nanoparticles released the chemicals and triggered gene expression in the plants under controlled-release conditions. Further developments such as pore enlargement and multifunctionalization of these MSNs may offer new possibilities in target-specific delivery of proteins, nucleotides and chemicals in plant biotechnology.

  1. Direct Nuclear Delivery of DNA by Photothermal Nanoblade.

    PubMed

    Wu, Ting-Hsiang; Wu, Yi-Chien; Sagullo, Enrico; Teitell, Michael A; Chiou, Pei-Yu

    2015-12-01

    We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed laser-triggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved. PMID:25900925

  2. HLA typing by direct DNA sequencing.

    PubMed

    Smith, Linda K

    2012-01-01

    Sequencing-based typing is a high resolution method for the identification of HLA polymorphisms. The majority of HLA Class I alleles can be discriminated by their exon 2 and 3 sequence, and for Class II alleles, exon 2 is generally sufficient. There are polymorphic positions in other exons which may require additional sequencing to exclude certain alleles with differences outside exon 2 and 3, depending on the clinical requirement and relevant accredition guidelines. The process involves selective amplification of target alleles by PCR, agarose gel electrophoresis of the PCR products to assess the quantity and quality, followed by purification of PCR amplicons to remove excess primer and dNTPs. Cycle sequencing reactions using Applied Biosystems™ BigDye(®) Terminator Ready Reaction v1.1 or v3.1 Kit are performed, then purification of sequence reactions before electrophoresing using Applied Biosystems™ 3730 or 3730XL Genetic Analyser (or similar). Data is processed by specialised software packages, which compare the sample sequence to the sequences of all possible theoretical allele combinations to assign an accurate genotype. Examination of all nucleotides, both at conserved and polymorphic positions enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing. PMID:22665229

  3. Direct-to-PCR tissue preservation for DNA profiling.

    PubMed

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition. PMID:26530406

  4. RNA-directed DNA methylation and demethylation in plants

    PubMed Central

    Viswanathan, CHINNUSAMY; Jian-Kang, ZHU

    2011-01-01

    RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit complementary siRNAs and the associated RdDM effector complex to the target loci in a transcription-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct target-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling enzymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Locus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses. PMID:19381459

  5. Toxicogenomic evaluation of chemically induced chromosomal imbalance using DNA image analysis.

    PubMed

    Hatzi, Vasiliki I; Terzoudi, Georgia I; Spiliopoulou, Chara A; Stefanidou, Maria E

    2013-06-01

    The study of carcinogenic potential of a variety of chemical agents such as food additives and drugs of abuse via the application of various in vitro methodologies constitutes the first step for the evaluation of their toxicogenomic profile. Considering the chromosomal theories of carcinogenesis, where it is stated that aneuploidy and chromosomal imbalance (instability) are among the main causes of carcinogenesis, chemicals capable to induce such changes in the cells could be considered as potential carcinogens. Chromosomal imbalance and aneuploidy directly affect the overall DNA content of the exposed cell as well as other cellular morpho- and densitometric features. These features can be measured by means of computerized DNA image analysis technologies and include DNA content (DNA Index), Proliferation Index, Ploidy Balance, Degree of Aneuploidy, Skewness and Kurtosis. Considering the enormous number of untested chemicals and drugs of abuse that follow non-genotoxic mechanisms of carcinogenesis, the establishment of a reliable technology for the estimation of chemically induced chromosomal imbalance is of particular importance in toxicogenomic studies. In the present article and based on our previously published work, we highlight the advantages of the applications of DNA image analysis technology in an easy-to-use experimental model for the evaluation of the potential risk of various chemicals. The use of this technology for the detection of chemically induced chromosomal instability will contribute to the development of safer regulatory directives concerning the use of chemicals in food and pharmaceutical industry, as well as in the clarification of mechanisms of action of drugs of abuse. PMID:23215871

  6. Direct nanomaterial-DNA contact effects on DNA and mutation induction.

    PubMed

    Thongkumkoon, P; Sangwijit, K; Chaiwong, C; Thongtem, S; Singjai, P; Yu, L D

    2014-04-01

    The toxicity of nanomaterials has been well known, but mechanisms involved have been little known. This study was aimed at looking at direct interaction between nanomaterials and naked DNA for some fundamental understanding. Two different types of nanomaterials, carbon nanotubes (CNTs) and tungsten trioxide (WO₃) nanoplates, were simply mixed with naked DNA plasmid, respectively, in two different contact modes, dry or wet (in solution), for varied time periods. DNA topological forms were analyzed for changes using gel electrophoresis and fluoro-spectrometry. The nanomaterial-contacted DNA was transferred into bacteria Escherichia coli (E. coli) cells for mutation observation. Certain types and degrees of DNA damage were observed, such as single strand break and double strand break, and bacterial mutation was confirmed. The DNA damage increased with the contacting time in an exponential manner and increased more rapidly in the initial stage for the wet contact. The nanomaterials-contacted DNA transferred bacteria had about less than 10% survival but almost 100% mutation for the surviving cells. The CNTs were more offensive than the metal oxide nanomaterials. The mutation spectrum from the DNA sequencing analysis showed that DNA point mutation was dominated by transversion, which was dominated by guanine changes in the wet contact condition while by cytosine changes in the dry contact condition. The point mutation occurrence in the wet contact was more than in the dry contact, confirming the wet contact more active and thus dangerous than dry contact. This experiment, although as a model study, revealed that direct simple contacts between nanomaterials and DNA could cause DNA changes and thus induce mutations which might potentially lead to cancers, diseases and genetic changes. This could be a mechanism for nanomaterial genotoxicity to the cells and also provided a caution to applications in using nanomaterials for DNA delivery. PMID:24503012

  7. siRNA-directed DNA Methylation in Plants

    PubMed Central

    Xie, Meng; Yu, Bin

    2015-01-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  8. Chemical Reactions Directed Peptide Self-Assembly

    PubMed Central

    Rasale, Dnyaneshwar B.; Das, Apurba K.

    2015-01-01

    Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly. PMID:25984603

  9. Direct Extraction and Amplification of DNA from Soil.

    ERIC Educational Resources Information Center

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  10. Chromosomal directionality of DNA mismatch repair in Escherichia coli

    PubMed Central

    Hasan, A. M. Mahedi; Leach, David R. F.

    2015-01-01

    Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5′ to 3′ and the 3′ to 5′ directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication. PMID:26170312

  11. Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

    PubMed Central

    Merkel, George; Alexandratos, Jerry; Zhou, Dongwen; Bojja, Ravi S.; Satoh, Tadashi; Potapov, Mikhail; Kogon, Alex; Potapov, Viktor; Wlodawer, Alexander; Skalka, Anna Marie

    2011-01-01

    Background We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. Methodology/Results Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. Conclusion Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs

  12. DNA damage induced by the direct effect of radiation

    NASA Astrophysics Data System (ADS)

    Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

    2008-10-01

    We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

  13. DNA Conjugation and DNA Directed Self-Assembly of Quantum Dots for Nanophotonic Applications

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban

    Colloidal quantum dots (QDs) or semiconductor nanocrystals are often used to describe 2--20 nm solution processed nanoparticles of various semiconductor materials that display quantum confinement effects. Compared to traditional fluorescent organic dyes, QDs provide many advantages. For biological applications it is necessary to develop reliable methods to functionalize QDs with hydrophilic biomolecules so that they may maintain their stability and functionality in physiological conditions. DNA, a molecule that encodes genetic information, is arguably the smartest molecule that nature has ever produced and one of the most explored bio-macromolecules. QDs that are functionalized with DNA can potentially be organized with nanometer precision by DNA directed self-assembly, and the resulting arrangements may facilitate the display of novel optical properties. The goal of this dissertation was to achieve a robust reliable yet simple strategy to link DNA to QDs so that they can be used for DNA directed self assembly by which we can engineer their optical properties. Presented here is a series of studies to achieve this goal. First we demonstrate the aqueous synthesis of colloidal nanocrystal heterostructures consisting of the CdTe core encapsulated by CdS/ZnS or CdSe/ZnS shells using glutathione (GSH), a tripeptide, as the capping ligand. We next employed this shell synthesis strategy to conjugate PS-PO chimeric DNA to QDs at the time of shell synthesis. We synthesized a library of DNA linked QDs emitting from UV to near IR that are very stable in high salt concentrations. These DNA functionalized QDs were further site-specifically organized on DNA origami in desired patterns directed by DNA self-assembly. We further extended our capability to functionalize DNA to real IR emitting CdxPb 1-xTe alloyed QDs, and demonstrated their stability by self-assembling them on DNA origami. The photo-physical properties of the QDs were further engineered by attaching a QD and a gold

  14. DNA barcoding to fishes: current status and future directions.

    PubMed

    Bhattacharya, Manojit; Sharma, Ashish Ranjan; Patra, Bidhan Chandra; Sharma, Garima; Seo, Eun-Min; Nam, Ju-Suk; Chakraborty, Chiranjib; Lee, Sang-Soo

    2016-07-01

    DNA barcoding appears to be a promising approach for taxonomic identification, characterization, and discovery of newer species, facilitating biodiversity studies. It helps researchers to appreciate genetic and evolutionary associations by collection of molecular, morphological, and distributional data. Fish DNA barcoding, based on the sequencing of a uniform area of Cytochrome C Oxidase type I (COI) gene, has received significant interest as an accurate tool for species identification, authentication, and phylogenetic analysis. The aim of this review article was to investigate recent global status, approaches, and future direction of DNA barcoding in fisheries sectors. We have tried to highlight its possible impacts, complications, and validation issues at species levels for biodiversity analysis. Moreover, an effort has been put forward to understand issues related to various marker genes associated with barcode process as primer sequences and have concluded barcode promotion as an indispensable tool of molecular biology for the development of taxonomic support systems. PMID:26057011

  15. DNA hybridization and ligation for directed colloidal assembly

    NASA Astrophysics Data System (ADS)

    Shyr, Margaret

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

  16. BIOASSAY-DIRECTED CHEMICAL ANALYSIS IN ENVIRONMENTAL RESEARCH

    EPA Science Inventory

    The use of short-term bioassay tests in conjunction with analytical measurements, constitute a powerful tool for identifying important environmental contaminants. The authors have coined the terminology 'bioassay directed chemical analysis' to best describe this marriage of analy...

  17. Directional imbibition on a chemically patterned silicon micropillar array.

    PubMed

    Jokinen, Ville

    2016-01-28

    Directional imbibition of oils (hexadecane, tetradecane, and dodecane) and water is demonstrated on a chemically patterned silicon micropillar array. Four different directional imbibition types are shown: unidirectional, two types of bidirectional and tridirectional imbibition. The surfaces consist of a silicon micropillar array with an overlaid surface chemistry pattern. This configuration leads to anisotropic wetting behaviour into various directions of the advancing meniscus. Due to the free energy landscape obtained, the advancing meniscus gets pinned in some directions (determined by the surface chemistry pattern) while it is free to move to the remaining directions. The conditions for directional imbibition and design criteria for the surfaces are derived and discussed. PMID:26576647

  18. A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.

    PubMed

    Pélissier, T; Wassenegger, M

    2000-01-01

    In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed. PMID:10668798

  19. Chemically amplified laser direct-writing of aluminum

    NASA Astrophysics Data System (ADS)

    Tsao, J. Y.; Ehrlich, D. J.

    Laser microchemical direct writing has important advantages over other techniques for the deposition of thin-film patterns. Disadvantages, however, are lower throughput and the need to suppress competing processes such as gas phase nucleation of particles or substrate damage. Methods for increasing the overall speed of laser direct writing by microchemistry were investigated. A class of laser deposition techniques has emerged in which laser radiation is used only to enhance or to impede the initial nucleation of a thin film. In general, it is convenient to draw a distinction between nucleation barriers due to physical effects and those due to chemical effects. The first type of barrier is derived from surface tension. The laser deposits a pattern of heterogeneous catalyst to initiate a subsequent transformation that is chemically self-sustaining or autocatalytic. Experiments, in which the laser direct writing of patterned thin films of Al is chemically amplified by subsequent selective pyrolytic chemical vapor deposition are summarized.

  20. Droplet heat transfer and chemical reactions during direct containment heating

    SciTech Connect

    Baker, L. Jr.

    1986-01-01

    A simplified model of heat transfer and chemical reaction has been adapted to evaluate the expected behavior of droplets containing unreacted Zircaloy and stainless steel moving through the containment atmosphere during postulated accidents involving direct containment heating. The model includes internal and external diffusive resistances to reaction. The results indicate that reactions will be incomplete for many conditions characteristic of direct containment heating sequences.

  1. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

    EPA Science Inventory

    One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

  2. Label free colorimetric and fluorimetric direct detection of methylated DNA based on silver nanoclusters for cancer early diagnosis.

    PubMed

    Dadmehr, Mehdi; Hosseini, Morteza; Hosseinkhani, Saman; Ganjali, Mohammad Reza; Sheikhnejad, Reza

    2015-11-15

    Epigenetic changes such as DNA methylation of CpG islands located in the promoter region of some tumor suppressor genes are very common in human diseases such as cancer. Detection of aberrant methylation pattern could serve as an excellent diagnostic approach. Recently, the direct detection of methylated DNA sequences without using chemical and enzymatic treatments or antibodies has received great deal of attentions. In this study, we report a colorimetric and fluorimetric technique for direct detection of DNA methylation. Here, the DNA is being used as an effective template for fluorescent silver nanoclusters formation without any chemical modification or DNA labeling. The sensitivity test showed that upon the addition of target methylated DNA, the fluorescence intensity is decreased in a linear range when the concentration of methylated DNA has increased from 2.0×10(-9) to 6.3 ×10(-7) M with the detection limit of 9.4×10(-10) M. The optical and fluorescence spectral behaviors were highly reproducible and clearly discriminated between unmethylated, methylated and even partially methylated DNA in CpG rich sequences. The results were also reproducible when the human plasma was present in our assay system. PMID:26056954

  3. DNA methylation directs functional maturation of pancreatic β cells

    PubMed Central

    Dhawan, Sangeeta; Tschen, Shuen-Ing; Zeng, Chun; Guo, Tingxia; Hebrok, Matthias; Matveyenko, Aleksey; Bhushan, Anil

    2015-01-01

    Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specific Dnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function. PMID:26098213

  4. Direct chemical evidence for eumelanin pigment from the Jurassic period.

    PubMed

    Glass, Keely; Ito, Shosuke; Wilby, Philip R; Sota, Takayuki; Nakamura, Atsushi; Bowers, C Russell; Vinther, Jakob; Dutta, Suryendu; Summons, Roger; Briggs, Derek E G; Wakamatsu, Kazumasa; Simon, John D

    2012-06-26

    Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms. PMID:22615359

  5. Direct chemical evidence for eumelanin pigment from the Jurassic period

    PubMed Central

    Glass, Keely; Ito, Shosuke; Wilby, Philip R.; Sota, Takayuki; Nakamura, Atsushi; Bowers, C. Russell; Vinther, Jakob; Dutta, Suryendu; Summons, Roger; Briggs, Derek E. G.; Wakamatsu, Kazumasa; Simon, John D.

    2012-01-01

    Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms. PMID:22615359

  6. Infrared emitting quantum dots: DNA conjugation and DNA origami directed self-assembly

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban; Deng, Zhengtao; Liu, Yan

    2014-04-01

    QDs that emit in the infrared (IR) range are of special interest at the moment because of their potential as tissue imaging reagents. Due to autofluorescence from tissues, QDs that emit in the visible range fail to produce good signal to noise ratios. Here we report the production of CdxPb1-xTe tertiary-alloyed QDs that emit in the 1100-1300 nm wavelength range, capped with the hydrophilic ligands mercaptopropionic acid (MPA) or glutathione (GSH), together with DNA, as specific surface tags. We observed an interesting dependence of the QD emission peaks on the species of capping ligand used. ICP-MS analysis confirmed that changing the identity of the surface ligand in the reaction mixture shifted the elemental composition of the particles and resulted in different Cd/Pb ratios. Further, DNA directed assembly of the particles onto DNA nanostructures ensures that the particle remains stable in high salt conditions, which is crucial to biological applications.QDs that emit in the infrared (IR) range are of special interest at the moment because of their potential as tissue imaging reagents. Due to autofluorescence from tissues, QDs that emit in the visible range fail to produce good signal to noise ratios. Here we report the production of CdxPb1-xTe tertiary-alloyed QDs that emit in the 1100-1300 nm wavelength range, capped with the hydrophilic ligands mercaptopropionic acid (MPA) or glutathione (GSH), together with DNA, as specific surface tags. We observed an interesting dependence of the QD emission peaks on the species of capping ligand used. ICP-MS analysis confirmed that changing the identity of the surface ligand in the reaction mixture shifted the elemental composition of the particles and resulted in different Cd/Pb ratios. Further, DNA directed assembly of the particles onto DNA nanostructures ensures that the particle remains stable in high salt conditions, which is crucial to biological applications. Electronic supplementary information (ESI) available

  7. Direct attachment of DNA to semiconducting surfaces for biosensor applications.

    PubMed

    Fahrenkopf, Nicholas M; Shahedipour-Sandvik, Fatemeh; Tokranova, Natalya; Bergkvist, Magnus; Cady, Nathaniel C

    2010-11-01

    In this work we propose a novel method of immobilizing nucleic acids for field effect or high electron mobility transistor-based biosensors. The naturally occurring 5' terminal phosphate group on nucleic acids was used to coordinate with semiconductor and metal oxide surfaces. We demonstrate that DNA can be directly immobilized onto ZrO(2), AlGaN, GaN, and HfO(2) while retaining its ability to hybridize to target sequences with high specificity. By directly immobilizing the probe molecule to the sensor surface, as opposed to conventional crosslinking strategies, the number of steps in device fabrication is reduced. Furthermore, hybridization to target strands occurs closer to the sensor surface, which has the potential to increase device sensitivity by reducing the impact of the Debye screening length. PMID:20869405

  8. Direct Measurements of Electrical Transport Through Single DNA Molecules of Complex Sequence

    NASA Astrophysics Data System (ADS)

    Cohen, Hezy; Nogues, Claude

    2005-03-01

    Seemingly contradicting results raised a debate over the ability of DNA to transport charge and the nature of the conduction mechanisms through it. We developed an experimental approach for measuring current through DNA molecules, chemically connected on opposite ends to a metal substrate and to a gold nanoparticle, using a conductive atomic force microscope.^1 Many samples could be made due to the experimental approach adopted here that enabled obtaining reproducible results in various samples, conditions and measurement methods. We present multileveled evidence for charge transport through 26 base-pairs long dsDNA of a complex sequence, characterized by S-shaped I-V curves that show currents higher than 220 nA at 2 V.^2 This significant observation implies that a coherent or band transport mechanism takes over for the high currents (> 1 nA). 1. Claude Nogues, Sidney R. Cohen, Shirley S. Daube, and Ron Naaman, ``Electrical properties of short DNA oligomers characterized by conducting atomic force microscopy,'' PCCP, 2004, 18. 2. Hezy Cohen, Claude Nogues Ron Naaman and Danny Porath. ``Direct Measurement of Electrical Transport Through Single DNA Molecules,'' submitted.

  9. DNA profiles from clothing fibers using direct PCR.

    PubMed

    Blackie, Renée; Taylor, Duncan; Linacre, Adrian

    2016-09-01

    We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number. PMID:27421265

  10. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    NASA Astrophysics Data System (ADS)

    Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

    2015-04-01

    The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

  11. Direct transfection of viral and plasmid DNA into the liver or spleen of mice.

    PubMed Central

    Dubensky, T W; Campbell, B A; Villarreal, L P

    1984-01-01

    A method for the direct transfection of polyoma viral DNA and polyoma-plasmid recombinant DNA into the liver or spleen of newborn or adult mice was developed. Calcium phosphate-precipitated DNA was injected directly into mouse organs in combination with hyaluronidase and collagenase. Transfected DNA was shown to replicate at moderate efficiency, relative to direct infection of organs with virus. Transfection with viral DNA rapidly led to an acute infection. A polyoma-bacterial plasmid recombinant DNA also was shown to replicate when transfected into mice. With this plasmid, however, genomic-length polyoma DNA rapidly recombined away from the bacterial component and replicated as viral DNA. This method should allow the direct determination of the biological activity of a cloned DNA within a mouse organ. Images PMID:6095303

  12. Bioassay-directed chemical analysis in environmental research

    SciTech Connect

    Schuetzle, D.; Lewtas, J.

    1986-01-01

    The use of short-term bioassay tests in conjunction with analytical measurements, constitute a powerful tool for identifying important environmental contaminants. The authors have coined the terminology bioassay directed chemical analysis to best describe this marriage of analytical chemistry and biology. The objective of this methodology is to identify key compounds in various types of air-pollutant samples. Once that task is completed, studies on metabolism, sources, environmental exposure and atmospheric chemistry can be undertaken. The principles and methodologies for bioassay directed chemical analysis are presented and illustrated in this paper. Most of this work has been directed toward the characterization of ambient air and diesel particulates, which are used as examples in this report to illustrate the analytical logic used for identifying the bio-active components of complex mixtures.

  13. Excision of plastid marker genes using directly repeated DNA sequences.

    PubMed

    Mudd, Elisabeth A; Madesis, Panagiotis; Avila, Elena Martin; Day, Anil

    2014-01-01

    Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. PMID:24599849

  14. Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

    PubMed

    Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2015-11-01

    Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. PMID:26447808

  15. Direct visualization of a DNA glycosylase searching for damage.

    PubMed

    Chen, Liwei; Haushalter, Karl A; Lieber, Charles M; Verdine, Gregory L

    2002-03-01

    DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases. PMID:11927259

  16. Large-area spatially ordered arrays of gold nanoparticles directed by lithographically confined DNA origami.

    PubMed

    Hung, Albert M; Micheel, Christine M; Bozano, Luisa D; Osterbur, Lucas W; Wallraff, Greg M; Cha, Jennifer N

    2010-02-01

    The development of nanoscale electronic and photonic devices will require a combination of the high throughput of lithographic patterning and the high resolution and chemical precision afforded by self-assembly. However, the incorporation of nanomaterials with dimensions of less than 10 nm into functional devices has been hindered by the disparity between their size and the 100 nm feature sizes that can be routinely generated by lithography. Biomolecules offer a bridge between the two size regimes, with sub-10 nm dimensions, synthetic flexibility and a capability for self-recognition. Here, we report the directed assembly of 5-nm gold particles into large-area, spatially ordered, two-dimensional arrays through the site-selective deposition of mesoscopic DNA origami onto lithographically patterned substrates and the precise binding of gold nanocrystals to each DNA structure. We show organization with registry both within an individual DNA template and between components on neighbouring DNA origami, expanding the generality of this method towards many types of patterns and sizes. PMID:20023644

  17. Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release?

    PubMed Central

    Razskazovskiy, Yuriy; Debije, Michael G.; Bernhard, William A.

    2008-01-01

    The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J−1. The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient. PMID:10761004

  18. A microsuspension adaptation of the Bacillus subtilis ''rec'' assay. [Detection of chemically induced DNA damage

    SciTech Connect

    McCarroll, N.E.; Keech, B.H.; Piper, C.E.

    1981-01-01

    We have demonstrated the utility of an Escherichia coli microsuspension assay to detect and characterize chemical mediation of DNA damage by a wide variety of mutagens and carcinogens. The assay have been improved by the development of a microsuspension modification to the Bacillus subtilis ''rec'' assay. The addition of these gram-positive organisms has allowed detection of DNA damage induced by benzo(a)pyrene (B(a)P), 3-aminopyrene (3-AP), 7, 12-dimethylbenz(a)anthrancene (DMBA), 3-methylcholanthrene (3-MC), and 4-nitrobiphenyl (4-NBP). Data presented in this paper from tests of 61 additional compounds, including a representative number of direct and promutagenic agents, indicate that the B subtilis H17 and M45 strains provide an effective microbial system for identification of DNA damage susceptible to postreplicational repair. The results of this study further suggests that the inclusion of these strains in the microsuspension assay for DNA damage will markedly enhance the detection of agents which cannot readily penetrate the intact cell wall of E coli.

  19. The direct peptide reactivity assay: selectivity of chemical respiratory allergens.

    PubMed

    Lalko, Jon F; Kimber, Ian; Gerberick, G Frank; Foertsch, Leslie M; Api, Anne Marie; Dearman, Rebecca J

    2012-10-01

    It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24 h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens. PMID:22713598

  20. Chemical Bonding Technology: Direct Investigation of Interfacial Bonds

    NASA Technical Reports Server (NTRS)

    Koenig, J. L.; Boerio, F. J.; Plueddemann, E. P.; Miller, J.; Willis, P. B.; Cuddihy, E. F.

    1986-01-01

    This is the third Flat-Plate Solar Array (FSA) Project document reporting on chemical bonding technology for terrestrial photovoltaic (PV) modules. The impetus for this work originated in the late 1970s when PV modules employing silicone encapsulation materials were undergoing delamination during outdoor exposure. At that time, manufacturers were not employing adhesion promoters and, hence, module interfaces in common with the silicone materials were only in physical contact and therefore easily prone to separation if, for example, water were to penetrate to the interfaces. Delamination with silicone materials virtually vanished when adhesion promoters, recommended by silicone manufacturers, were used. The activities related to the direct investigation of chemically bonded interfaces are described.

  1. Direct Evidence for the Formation of Precatenanes during DNA Replication*

    PubMed Central

    Cebrián, Jorge; Castán, Alicia; Martínez, Víctor; Kadomatsu-Hermosa, Maridian J.; Parra, Cristina; Fernández-Nestosa, María José; Schaerer, Christian; Hernández, Pablo; Krimer, Dora B.; Schvartzman, Jorge B.

    2015-01-01

    The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination. PMID:25829493

  2. Brownian dynamics simulation of directional sliding of histone octamers caused by DNA bending

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2006-05-01

    Chromatin-remodeling complexes such as SWI/SNF and RSC of yeast can perturb the structure of nucleosomes in an ATP-dependent manner. Experimental results prove that this chromatin remodeling process involves DNA bending. We simulate the effect of DNA bending, caused by chromatin-remodeling complexes, on directional sliding of histone octamers by Brownian dynamics simulation. The simulation results show that, after a DNA loop being generated at the side of a nucleosome, the histone octamer slides towards this DNA loop until the loop disappears. The DNA loop size is an important factor affecting the process of directional sliding of the histone octamer.

  3. Directed organization of DNA filaments in a soft matter template.

    PubMed

    De Sio, Luciano; D'Aquila, Patrizia; Brunelli, Elvira; Strangi, Giuseppe; Bellizzi, Dina; Passarino, Giuseppe; Umeton, Cesare; Bartolino, Roberto

    2013-03-12

    We have developed a noninvasive, all-optical, holographic technique for permanently aligning liquid crystalline DNA filaments in a microperiodic template realized in soft-composite (polymeric) materials. By combining optical intensity holography with a selective microfluidic etching process, a channelled microstructure has been realized which enables self-assembly of DNA. The striking chemicophysical properties of the structure immobilize the DNA filaments within the microchannels without the need of any kind of surface chemistry or functionalization. Polarized optical, confocal, and electronic microscopies have been used for characterizing the DNA geometry inside the microchannels in terms of birefringence, fluorescence, and nanoscale organization properties. In particular, observation of a far-field diffraction pattern confirms a periodic organization of the DNA filaments inside the polymeric template. PMID:23425153

  4. Comparison of six commercially-available DNA polymerases for direct PCR.

    PubMed

    Miura, Masashi; Tanigawa, Chihiro; Fujii, Yoshito; Kaneko, Satoshi

    2013-01-01

    The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. PMID:24213192

  5. {beta}-carboline derivatives: Novel photosensitizers that intercalate into DNA to cause direct DNA damage in photodynamic therapy

    SciTech Connect

    Guan Huaji; Liu Xiaodong; Peng Wenlie; Cao Rihui; Ma Yan; Chen Hongsheng; Xu Anlong . E-mail: ls36@zsu.edu.cn

    2006-04-14

    Novel 1,3,9-trisubstituted {beta}-carboline derivatives were found to exhibit DNA photocleavage properties under visible light irradiation in a cell-free system, which could be reduced by antioxidant vitamin E. Their photo-cytotoxicity to human tumor cell line HeLa was confirmed, in which apoptosis only contributed a small part to the cell death, and necrosis was the dominating outcome of HeLa cells in photodynamic therapy (PDT) using {beta}-carboline derivatives. Different from other clinical PDT drugs, {beta}-carboline derivatives were demonstrated to be able to distribute in the nucleus and intercalate into DNA, and consequently cause direct DNA damage by photochemical reaction products in PDT, which was proved by the distinct DNA tails in the comet assay and the considerable amount of DNA damaged cells quantified by flow cytometry. This mechanism could be the explanation for the delay of cell proliferation at DNA synthesis and mitosis.

  6. Direct chemical oxidation of mixed or toxic wastes

    SciTech Connect

    Balazs, G B; Cooper, J F; Farmer, J C; Lewis, P

    1999-05-01

    Direct Chemical Oxidation (DCO) is an ambient-pressure, low-temperature (<100 C), and aqueous-based process for general-purpose destruction of the organic fraction of hazardous or mixed waste. It uses the peroxydisulfate anion (S{sub 2}O{sub 8}{sup 2{minus}}) in acid or base solutions. The byproduct of the oxidation reaction, typically sodium or ammonium hydrogen sulfate, may be recycled electrolytically to produce the oxidant. The oxidation kinetic reaction is first order with respect to the peroxydisulfate concentration, expressed in equivalents. The rate constant is constant for nearly all dissolved organic compounds: k{sub a} = 0.01 {+-} 0.005 min{sup {minus}1}. This reflects a common rate-determining step, which is the decomposition of the peroxydisulfate anion into the chemically active derivative, the sulfate radical anion, SO{sub 4}{sup {minus}}. This decomposition is promoted in DCO by raising the operating temperature into the range of 80-100 C. Rates are given for approximately 30 substances with diverse functional groups at low concentrations, and for a number of solid and liquid wastes typical of nuclear and chemical industries. The process has been scale up for treatment studies on chlorinated hydrocarbons, in which the hydrolysis of solvent mixtures was followed by oxidation of products in a series of stirred tank reactors. Cost estimates, safety considerations, and a comprehensive bibliography are given.

  7. Direct simulations of chemically reacting turbulent mixing layers, part 2

    NASA Technical Reports Server (NTRS)

    Metcalfe, Ralph W.; Mcmurtry, Patrick A.; Jou, Wen-Huei; Riley, James J.; Givi, Peyman

    1988-01-01

    The results of direct numerical simulations of chemically reacting turbulent mixing layers are presented. This is an extension of earlier work to a more detailed study of previous three dimensional simulations of cold reacting flows plus the development, validation, and use of codes to simulate chemically reacting shear layers with heat release. Additional analysis of earlier simulations showed good agreement with self similarity theory and laboratory data. Simulations with a two dimensional code including the effects of heat release showed that the rate of chemical product formation, the thickness of the mixing layer, and the amount of mass entrained into the layer all decrease with increasing rates of heat release. Subsequent three dimensional simulations showed similar behavior, in agreement with laboratory observations. Baroclinic torques and thermal expansion in the mixing layer were found to produce changes in the flame vortex structure that act to diffuse the pairing vortices, resulting in a net reduction in vorticity. Previously unexplained anomalies observed in the mean velocity profiles of reacting jets and mixing layers were shown to result from vorticity generation by baroclinic torques.

  8. Direct simulations of chemically reacting turbulent mixing layers, part 2

    NASA Astrophysics Data System (ADS)

    Metcalfe, Ralph W.; McMurtry, Patrick A.; Jou, Wen-Huei; Riley, James J.; Givi, Peyman

    1988-06-01

    The results of direct numerical simulations of chemically reacting turbulent mixing layers are presented. This is an extension of earlier work to a more detailed study of previous three dimensional simulations of cold reacting flows plus the development, validation, and use of codes to simulate chemically reacting shear layers with heat release. Additional analysis of earlier simulations showed good agreement with self similarity theory and laboratory data. Simulations with a two dimensional code including the effects of heat release showed that the rate of chemical product formation, the thickness of the mixing layer, and the amount of mass entrained into the layer all decrease with increasing rates of heat release. Subsequent three dimensional simulations showed similar behavior, in agreement with laboratory observations. Baroclinic torques and thermal expansion in the mixing layer were found to produce changes in the flame vortex structure that act to diffuse the pairing vortices, resulting in a net reduction in vorticity. Previously unexplained anomalies observed in the mean velocity profiles of reacting jets and mixing layers were shown to result from vorticity generation by baroclinic torques.

  9. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  10. Direct observation of DNA overwinding by reverse gyrase

    PubMed Central

    Ogawa, Taisaku; Yogo, Katsunori; Furuike, Shou; Sutoh, Kazuo; Kikuchi, Akihiko; Kinosita, Kazuhiko

    2015-01-01

    Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 °C. The unloaded reaction rate at 71 °C exceeds five turns per second, which is >102-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s−1, indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5 pN⋅nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding. PMID:26023188

  11. Mechano-chemical selections of two competitive unfolding pathways of a single DNA i-motif

    NASA Astrophysics Data System (ADS)

    Xu, Yue; Chen, Hu; Qu, Yu-Jie; Artem, K. Efremov; Li, Ming; Ouyang, Zhong-Can; Liu, Dong-Sheng; Yan, Jie

    2014-06-01

    The DNA i-motif is a quadruplex structure formed in tandem cytosine-rich sequences in slightly acidic conditions. Besides being considered as a building block of DNA nano-devices, it may also play potential roles in regulating chromosome stability and gene transcriptions. The stability of i-motif is crucial for these functions. In this work, we investigated the mechanical stability of a single i-motif formed in the human telomeric sequence 5'-(CCCTAA)3CCC, which revealed a novel pH and loading rate-dependent bimodal unfolding force distribution. Although the cause of the bimodal unfolding force species is not clear, we proposed a phenomenological model involving a direct unfolding favored at lower loading rate or higher pH value, which is subject to competition with another unfolding pathway through a mechanically stable intermediate state whose nature is yet to be determined. Overall, the unique mechano—chemical responses of i-motif-provide a new perspective to its stability, which may be useful to guide designing new i-motif-based DNA mechanical nano-devices.

  12. Evolution of DNA and RNA as catalysts for chemical reactions.

    PubMed

    Jäschke, A; Seelig, B

    2000-06-01

    In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA molecules that have catalytic properties. Catalyzed reactions now go far beyond self-modifying reactions of nucleic acid molecules. The future application of in vitro selected RNA and DNA catalysts in bioorganic synthesis appears promising. PMID:10826969

  13. Fabrication of Ultrasensitive Field-Effect Transistor DNA Biosensors by a Directional Transfer Technique Based on CVD-Grown Graphene.

    PubMed

    Zheng, Chao; Huang, Le; Zhang, Hong; Sun, Zhongyue; Zhang, Zhiyong; Zhang, Guo-Jun

    2015-08-12

    Most graphene field-effect transistor (G-FET) biosensors are fabricated through a routine process, in which graphene is transferred onto a Si/SiO2 substrate and then devices are subsequently produced by micromanufacture processes. However, such a fabrication approach can introduce contamination onto the graphene surface during the lithographic process, resulting in interference for the subsequent biosensing. In this work, we have developed a novel directional transfer technique to fabricate G-FET biosensors based on chemical-vapor-deposition- (CVD-) grown single-layer graphene (SLG) and applied this biosensor for the sensitive detection of DNA. A FET device with six individual array sensors was first fabricated, and SLG obtained by the CVD-growth method was transferred onto the sensor surface in a directional manner. Afterward, peptide nucleic acid (PNA) was covalently immobilized on the graphene surface, and DNA detection was realized by applying specific target DNA to the PNA-functionalized G-FET biosensor. The developed G-FET biosensor was able to detect target DNA at concentrations as low as 10 fM, which is 1 order of magnitude lower than those reported in a previous work. In addition, the biosensor was capable of distinguishing the complementary DNA from one-base-mismatched DNA and noncomplementary DNA. The directional transfer technique for the fabrication of G-FET biosensors is simple, and the as-constructed G-FET DNA biosensor shows ultrasensitivity and high specificity, indicating its potential application in disease diagnostics as a point-of-care tool. PMID:26203889

  14. Construction of a DNA Nano-Object Directly Demonstrates Computation

    PubMed Central

    Wu, Gang; Jonoska, Natasha; Seeman, Nadrian C.

    2009-01-01

    We demonstrate a computing method in which a DNA nano-object representing the solution of a problem emerges as a result of self-assembly. We report an experiment in which three-vertex colorability for a 6-vertex graph with 9 edges is solved by constructing a DNA molecule representing the colored graph itself. Our findings show that computation based on “shape processing” is a viable alternative to symbol processing when computing by molecular self-assembly. PMID:19607875

  15. Direct measurement of the dielectric polarization properties of DNA

    PubMed Central

    Cuervo, Ana; Dans, Pablo D.; Carrascosa, José L.; Orozco, Modesto; Gomila, Gabriel; Fumagalli, Laura

    2014-01-01

    The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2–4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques. PMID:25136104

  16. Direct measurement of the dielectric polarization properties of DNA.

    PubMed

    Cuervo, Ana; Dans, Pablo D; Carrascosa, José L; Orozco, Modesto; Gomila, Gabriel; Fumagalli, Laura

    2014-09-01

    The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2-4), we found that the DNA dielectric constant is ∼ 8, considerably higher than the value of ∼ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson-Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques. PMID:25136104

  17. Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".

    PubMed

    Petric, I; Philippot, L; Abbate, C; Bispo, A; Chesnot, T; Hallin, S; Laval, K; Lebeau, T; Lemanceau, P; Leyval, C; Lindström, K; Pandard, P; Romero, E; Sarr, A; Schloter, M; Simonet, P; Smalla, K; Wilke, B-M; Martin-Laurent, F

    2011-03-01

    Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring. PMID:21256879

  18. DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging.

    PubMed

    Bombera, Radoslaw; Leroy, Loïc; Livache, Thierry; Roupioz, Yoann

    2012-03-15

    Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features. PMID:22236780

  19. The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

    SciTech Connect

    Stella, Stefano; Cascio, Duilio; Johnson, Reid C.

    2010-06-21

    The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of {approx}65{sup o}, and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

  20. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

    PubMed

    Ngo, Thuy T M; Zhang, Qiucen; Zhou, Ruobo; Yodh, Jaya G; Ha, Taekjip

    2015-03-12

    Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications. PMID:25768909

  1. Asymmetric Unwrapping of Nucleosomes under Tension Directed by DNA Local Flexibility

    PubMed Central

    Ngo, Thuy T. M.; Zhang, Qiucen; Zhou, Ruobo

    2015-01-01

    Summary Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications. PMID:25768909

  2. Chemical carcinogenesis — the price for DNA-repair?

    NASA Astrophysics Data System (ADS)

    Wintersberger, Ulrike

    1982-03-01

    This essay examines the possibility of merging the mutation theory of cancer with the hypothesis that cancer is a change in the state of the differentiation of cells. It is suggested that during normal development DNA rearrangements occur, concerning genes which code for differentiation specific cell communication proteins. These proteins are responsible for the proper functioning of growth control in a multicellular organism. DNA-damaging agents — mutagens — induce DNA repair enzymes, some of which may catalyse illegitimate genome rearrangements, thus leading to a change of the balance between growth and differentiation. A cell with a selective advantage may arise and become the origin of a tumor.

  3. New approach for direct chemical synthesis of hexagonal Co nanoparticles

    NASA Astrophysics Data System (ADS)

    Abel, Frank M.; Tzitzios, Vasilis; Hadjipanayis, George C.

    2016-02-01

    In this paper, we explore the possibility of producing hexagonal Cobalt nanoparticles, with high saturation magnetization by direct chemical synthesis. The nanoparticles were synthesized by reduction of anhydrous cobalt (II) chloride by NaBH4 in tetraglyme at temperatures in the range of 200-270 °C under a nitrogen-hydrogen atmosphere. The reactions were done at high temperatures to allow for the formation of as-made hexagonal cobalt. The size of the particles was controlled by the addition of different surfactants. The best magnetic properties so far were obtained on spherical hexagonal Co nanoparticles with an average size of 45 nm, a saturation magnetization of 143 emu/g and coercivity of 500 Oe. the saturation magnetization and coercivity were further improved by annealing the Co nanoparticles leading to saturation magnetization of 160 emu/g and coercivity of 540 Oe.

  4. Characterization of glycosphingolipids by direct inlet chemical ionization mass spectrometry.

    PubMed

    Ariga, T; Murata, T; Oshima, M; Maezawa, M; Miyatake, T

    1980-09-01

    Permethylated derivatives of cerebrosides and ceramide di-, tri-, tetra-, and penta-hexosides were analyzed by the direct inlet ammonia chemical ionization (CI) mass spectrometry. In the CI mass spectra, the fragment ions produced by the loss of methanol from the protonated molecular ion were observed in all of the glycosphingolipids. Other fragment ions due to the cleavage of glycosidic moiety were major ones under the CI conditions. These ions provide information on the molecular species of glycosphingolipids and the sugar sequence of their oligosaccharides. Glycosphingolipids with hydroxy fatty acids could also be differentiated from those with nonhydroxy fatty acids by comparing the intensities of characteristic fragment ions. The CI method should be particularly useful in structural studies of glycosphingolipids from natural sources. PMID:7441059

  5. Direct simulations of chemically reacting turbulent mixing layers

    NASA Technical Reports Server (NTRS)

    Riley, J. J.; Metcalfe, R. W.

    1984-01-01

    The report presents the results of direct numerical simulations of chemically reacting turbulent mixing layers. The work consists of two parts: (1) the development and testing of a spectral numerical computer code that treats the diffusion reaction equations; and (2) the simulation of a series of cases of chemical reactions occurring on mixing layers. The reaction considered is a binary, irreversible reaction with no heat release. The reacting species are nonpremixed. The results of the numerical tests indicate that the high accuracy of the spectral methods observed for rigid body rotation are also obtained when diffusion, reaction, and more complex flows are considered. In the simulations, the effects of vortex rollup and smaller scale turbulence on the overall reaction rates are investigated. The simulation results are found to be in approximate agreement with similarity theory. Comparisons of simulation results with certain modeling hypotheses indicate limitations in these hypotheses. The nondimensional product thickness computed from the simulations is compared with laboratory values and is found to be in reasonable agreement, especially since there are no adjustable constants in the method.

  6. Delay chemical master equation: direct and closed-form solutions

    PubMed Central

    Leier, Andre; Marquez-Lago, Tatiana T.

    2015-01-01

    The stochastic simulation algorithm (SSA) describes the time evolution of a discrete nonlinear Markov process. This stochastic process has a probability density function that is the solution of a differential equation, commonly known as the chemical master equation (CME) or forward-Kolmogorov equation. In the same way that the CME gives rise to the SSA, and trajectories of the latter are exact with respect to the former, trajectories obtained from a delay SSA are exact representations of the underlying delay CME (DCME). However, in contrast to the CME, no closed-form solutions have so far been derived for any kind of DCME. In this paper, we describe for the first time direct and closed solutions of the DCME for simple reaction schemes, such as a single-delayed unimolecular reaction as well as chemical reactions for transcription and translation with delayed mRNA maturation. We also discuss the conditions that have to be met such that such solutions can be derived. PMID:26345616

  7. Proton-induced direct and indirect damage of plasmid DNA.

    PubMed

    Vyšín, Luděk; Pachnerová Brabcová, Kateřina; Štěpán, Václav; Moretto-Capelle, Patrick; Bugler, Beatrix; Legube, Gaelle; Cafarelli, Pierre; Casta, Romain; Champeaux, Jean Philippe; Sence, Martine; Vlk, Martin; Wagner, Richard; Štursa, Jan; Zach, Václav; Incerti, Sebastien; Juha, Libor; Davídková, Marie

    2015-08-01

    Clustered DNA damage induced by 10, 20 and 30 MeV protons in pBR322 plasmid DNA was investigated. Besides determination of strand breaks, additional lesions were detected using base excision repair enzymes. The plasmid was irradiated in dry form, where indirect radiation effects were almost fully suppressed, and in water solution containing only minimal residual radical scavenger. Simultaneous irradiation of the plasmid DNA in the dry form and in the solution demonstrated the contribution of the indirect effect as prevalent. The damage composition slightly differed when comparing the results for liquid and dry samples. The obtained data were also subjected to analysis concerning different methodological approaches, particularly the influence of irradiation geometry, models used for calculation of strand break yields and interpretation of the strand breaks detected with the enzymes. It was shown that these parameters strongly affect the results. PMID:26007308

  8. Directed assembly of discrete gold nanoparticle groupings usingbranched DNA scaffolds

    SciTech Connect

    Claridge, Shelley A.; Goh, Sarah L.; Frechet, Jean M.J.; Williams, Shara C.; Micheel, Christine M.; Alivisatos, A. Paul

    2004-09-14

    The concept of self-assembled dendrimers is explored for the creation of discrete nanoparticle assemblies. Hybridization of branched DNA trimers and nanoparticle-DNA conjugates results in the synthesis of nanoparticle trimer and tetramer complexes. Multiple tetramer architectures are investigated, utilizing Au-DNA conjugates with varying secondary structural motifs. Hybridization products are analyzed by gel electrophoresis, and discrete bands are observed corresponding to structures with increasing numbers of hybridization events. Samples extracted from each band are analyzed by transmission electron microscopy, and statistics compiled from micrographs are used to compare assembly characteristics for each architecture. Asymmetric structures are also produced in which both 5 and 10 nm Au particles are assembled on branched scaffolds.

  9. Increment of DNA topoisomerases in chemically and virally transformed cells

    SciTech Connect

    Crespi, M.D.; Mladovan, A.G.; Baldi, A. )

    1988-03-01

    The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo(a)pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between {sup 32}P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo(a)pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.

  10. Theoretical studies of chemical reactivity of metabolically activated forms of aromatic amines toward DNA.

    PubMed

    Shamovsky, Igor; Ripa, Lena; Blomberg, Niklas; Eriksson, Leif A; Hansen, Peter; Mee, Christine; Tyrchan, Christian; O'Donovan, Mike; Sjö, Peter

    2012-10-15

    The metabolism of aromatic and heteroaromatic amines (ArNH₂) results in nitrenium ions (ArNH⁺) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH⁺ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH₂, including the majority of known genotoxic carcinogens, the stability of ArNH⁺ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH⁺ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH₂, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH⁺ accelerates with resonance stabilization of ArNH⁺, whereas the formed ArNH⁺ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH⁺ is about 20 kcal/mol more stable than PhNH⁺. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH⁺ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (π-TS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH⁺. We demonstrate that the mutagenic potency of ArNH₂ correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH₂. The mechanism-based reasoning suggests rational design strategies to

  11. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES

    EPA Science Inventory

    A simple and rapid assay to detect DNA damage is reported. This assay is based on the ability of certain dyes to fluoresce upon intercalation with dsDNA. Damage caused by ultraviolet (UV) radiation, chemicals or restriction enzymes is detected using this assay. UV radiation at...

  12. DEVELOPMENT OF RAPID TECHNIQUES FOR DETECTION OF CHEMICALLY-INDUCED DNA DAMAGE

    EPA Science Inventory

    Rapid and cost-effective indicator assays are being developed which may be used as a rapid screen to assess the potential for exposure to hazardous compounds that can be related to a biological target (e.g., DNA). Chemically-induced DNA damage will be measured using surrogate DN...

  13. The direct application of the polymerase chain reaction to DNA extracted from foods.

    PubMed

    Dickinson, J H; Kroll, R G; Grant, K A

    1995-04-01

    Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods. PMID:7766115

  14. DNA binding fluorescent proteins for the direct visualization of large DNA molecules.

    PubMed

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely 'paints' entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  15. DNA binding fluorescent proteins for the direct visualization of large DNA molecules

    PubMed Central

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C.

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  16. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2015-01-01

    During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN). PMID:25800740

  17. Chemically directed assembly of nanoparticles for material and biological applications

    NASA Astrophysics Data System (ADS)

    Park, Myoung-Hwan

    The unique electronic, magnetic, and optical properties of nanoparticles (NPs) make them useful building blocks for nanodevices and biofabrication. Site-selective immobilization/deposition of NPs on surfaces at desired positions is an important fabrication step in realizing the potential of nanomaterials in these applications. In this thesis, my research has focused on developing new strategies for mono- and multilayered-NP deposition on surfaces, increasing the stability of NP-assembles upon various surfaces for practical use of NP-based devices. Chemically directed dithiocarbamate binding of amine groups to NPs in the presence of CS2 was used for enhancing the robustness of NP assembles. Such patterning methodologies have allowed me to use site-directed NP immobilization in applications as diverse as microcontact printing, nanomolding in capillaries, nanoimprint lithography, and photolithography. Also, I have developed a simple and reliable one-step technique to form robust dendrimer-NP nanocomposites using dithiocarbamate-based chemistry. These composites are able to encapsulate and release various therapeutics, providing controllable sustained release and to separate small molecules and biomacromolecules.

  18. Direct observation of DNA threading in flap endonuclease complexes.

    PubMed

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

    2016-07-01

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

  19. Direct interaction between cohesin complex and DNA replication machinery

    SciTech Connect

    Ryu, Min-Jung; Kim, Beom-Jun; Lee, Jeong-Won; Lee, Min-Woo; Choi, Hyun-Kyung; Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2006-03-17

    Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase {alpha}. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.

  20. The Influence of DNA Configuration on the Direct Strand Break Yield

    PubMed Central

    Bernal, M. A.; deAlmeida, C. E.; Incerti, S.; Champion, C.; Ivanchenko, V.; Francis, Z.

    2015-01-01

    Purpose. To study the influence of DNA configuration on the direct damage yield. No indirect effect has been accounted for. Methods. The GEANT4-DNA code was used to simulate the interactions of protons and alpha particles with geometrical models of the A-, B-, and Z-DNA configurations. The direct total, single, and double strand break yields and site-hit probabilities were determined. Certain features of the energy deposition process were also studied. Results. A slight increase of the site-hit probability as a function of the incident particle linear energy transfer was found for each DNA configuration. Each DNA form presents a well-defined site-hit probability, independently of the particle linear energy transfer. Approximately 70% of the inelastic collisions and ~60% of the absorbed dose are due to secondary electrons. These fractions are slightly higher for protons than for alpha particles at the same incident energy. Conclusions. The total direct strand break yield for a given DNA form depends weakly on DNA conformation topology. This yield is practically determined by the target volume of the DNA configuration. However, the double strand break yield increases with the packing ratio of the DNA double helix; thus, it depends on the DNA conformation. PMID:26124855

  1. Direct measurement of DNA-mediated adhesion between lipid bilayers.

    PubMed

    Shimobayashi, S F; Mognetti, B M; Parolini, L; Orsi, D; Cicuta, P; Di Michele, L

    2015-06-28

    Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates. Lately, analogous artificial materials have been synthesised by functionalising the outer surface of compliant Brownian units, for example emulsion droplets and lipid vesicles, with selective linkers, in particular short DNA sequences. This development extended the range of applicability of DNA as a selective glue, originally applied to solid nano and colloidal particles. On very deformable lipid vesicles, the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours, as we recently contributed to demonstrate [Parolini et al., Nat. Commun., 2015, 6, 5948]. Several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and a fundamental understanding. Here we focus on the DNA-mediated multivalent interactions of a single liposome adhering to a flat supported bilayer. This simplified geometry enables the estimate of the membrane tension induced by the DNA-mediated adhesive forces acting on the liposome. Our experimental investigation is completed by morphological measurements and the characterisation of the DNA-melting transition, probed by in situ Förster Resonant Energy Transfer spectroscopy. Experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile linkers. With at most one fitting parameter, our theory is capable of semi-quantitatively matching experimental data, confirming the quality of the underlying assumptions. PMID:25989828

  2. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  3. DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

    PubMed Central

    Riazuddin, S; Athar, A; Ahmed, Z; Lali, S M; Sohail, A

    1987-01-01

    Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively. PMID:3628000

  4. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

    PubMed Central

    2016-01-01

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design. PMID:25853314

  5. Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

    PubMed Central

    Chen, Yuan-Jyue; Rao, Sundipta D.; Seelig, Georg

    2015-01-01

    DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA. PMID:26649734

  6. Physisorption of DNA molecules on chemically modified single-walled carbon nanotubes with and without sonication.

    PubMed

    Umemura, Kazuo; Ishibashi, Yu; Oura, Shusuke

    2016-09-01

    We investigated the physisorption phenomenon of single-stranded DNA (ssDNA) molecules onto two types of commercially available chemically functionalized single-walled carbon nanotubes (SWNTs) by atomic force microscopy (AFM) and agarose gel electrophoresis. We found that DNA molecules can adsorb on the water-soluble SWNT surfaces without sonication, although sonication treatment has been used for hybridization of DNA and SWNTs in many previous studies. Using our method, damage of DNA molecules by sonication can be avoided. On the other hand, the amount of DNA molecules adsorbed on SWNT surfaces increased when the samples were sonicated. This fact suggests that the sonication is effective not only at debundling of SWNTs, but also at assisting DNA adsorption. Furthermore, DNA adsorption was affected by the types of functionalized SWNTs. In the case of SWNTs functionalized with polyethylene glycol (PEG-SWNT), physisorption of ssDNA molecules was confirmed only by agarose-gel electrophoresis. In contrast, amino-terminated SWNTs (NH2-SWNTs) showed a change in the height distribution profile based on AFM observations. These results suggest that DNA molecules tended to adsorb to NH2-SWNT surfaces, although DNA molecules can also adsorb on PEG-SWNT surfaces. Our results revealed fundamental information for developing nanobiodevices using hybrids of DNA and SWNTs. PMID:26846296

  7. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  8. Direct imaging of single UvrD helicase dynamics on long single-stranded DNA.

    PubMed

    Lee, Kyung Suk; Balci, Hamza; Jia, Haifeng; Lohman, Timothy M; Ha, Taekjip

    2013-01-01

    Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding. PMID:23695672

  9. Directed enzymatic activation of 1-D DNA tiles.

    PubMed

    Garg, Sudhanshu; Chandran, Harish; Gopalkrishnan, Nikhil; LaBean, Thomas H; Reif, John

    2015-02-24

    The tile assembly model is a Turing universal model of self-assembly where a set of square shaped tiles with programmable sticky sides undergo coordinated self-assembly to form arbitrary shapes, thereby computing arbitrary functions. Activatable tiles are a theoretical extension to the Tile assembly model that enhances its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly. In this article, we experimentally demonstrate a simplified version of the Activatable tile assembly model. In particular, we demonstrate the simultaneous assembly of protected DNA tiles where a set of inert tiles are activated via a DNA polymerase to undergo linear assembly. We then demonstrate stepwise activated assembly where a set of inert tiles are activated sequentially one after another as a result of attachment to a growing 1-D assembly. We hope that these results will pave the way for more sophisticated demonstrations of activated assemblies. PMID:25625898

  10. DNA directed self-assembly of shape-controlled hydrogels

    PubMed Central

    Qi, Hao; Ghodousi, Majid; Du, Yanan; Grun, Casey; Bae, Hojae; Yin, Peng; Khademhosseini, Ali

    2013-01-01

    Using DNA as programmable, sequence specific ‘glues’, shape-controlled hydrogel units are self-assembled into prescribed structures. Here we report that aggregates are produced using hydrogel cubes with edge length ranging from 30 micrometers to 1 millimeter, demonstrating assembly across scales. In a simple one-pot agitation reaction, 25 dimers are constructed in parallel from 50 distinct hydrogel cube species, demonstrating highly multiplexed assembly. Using hydrogel cuboids displaying face-specific DNA glues, diverse structures are achieved in aqueous and in interfacial agitation systems. These include dimers, extended chains, and open network structures in an aqueous system; and dimers, chains of fixed length, T-junctions, and square shapes in the interfacial system, demonstrating the versatility of the assembly system. PMID:24013352

  11. Direct detection of DNA conformation in hybridization processes.

    PubMed

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

    2012-02-21

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

  12. Mapping site-specific endonuclease binding to DNA by direct imaging with AFM

    SciTech Connect

    Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P.; Isfort, R.J.

    1995-12-31

    Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in the preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

  13. Liposome-based chemical barcodes for single molecule DNA detection using imaging mass spectrometry.

    PubMed

    Gunnarsson, Anders; Sjövall, Peter; Höök, Fredrik

    2010-02-10

    We report on a mass-spectrometry (time-of-flight secondary ion mass spectrometry, TOF-SIMS) based method for multiplexed DNA detection utilizing a random array, where the lipid composition of small unilamellar liposomes act as chemical barcodes to identify unique DNA target sequences down to the single molecule level. In a sandwich format, suspended target-DNA to be detected mediates the binding of capture-DNA modified liposomes to surface-immobilized probe-DNA. With the lipid composition of each liposome encoding a unique target-DNA sequence, TOF-SIMS analysis was used to determine the chemical fingerprint of the bound liposomes. Using high-resolution TOF-SIMS imaging, providing sub-200 nm spatial resolution, single DNA targets could be detected and identified via the chemical fingerprint of individual liposomes. The results also demonstrate the capability of TOF-SIMS to provide multiplexed detection of DNA targets on substrate areas in the micrometer range. Together with a high multiplexing capacity, this makes the concept an interesting alternative to existing barcode concepts based on fluorescence, Raman, or graphical codes for small-scale bioanalysis. PMID:20085369

  14. Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

    PubMed Central

    Kulczyk, Arkadiusz W.; Tanner, Nathan A.; Loparo, Joseph J.; Richardson, Charles C.; van Oijen, Antoine M.

    2010-01-01

    We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2). PMID:20332766

  15. RecG Directs DNA Synthesis during Double-Strand Break Repair

    PubMed Central

    Azeroglu, Benura; Mawer, Julia S. P.; Cockram, Charlotte A.; White, Martin A.; Hasan, A. M. Mahedi; Filatenkova, Milana; Leach, David R. F.

    2016-01-01

    Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability. PMID:26872352

  16. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.

    PubMed

    Lucius, Aaron L; Vindigni, Alessandro; Gregorian, Razmic; Ali, Janid A; Taylor, Andrew F; Smith, Gerald R; Lohman, Timothy M

    2002-11-29

    The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding. PMID:12445778

  17. Physical controls on directed virus assembly at nanoscale chemical templates

    SciTech Connect

    Cheung, C L; Chung, S; Chatterji, A; Lin, T; Johnson, J E; Hok, S; Perkins, J; De Yoreo, J

    2006-05-10

    Viruses are attractive building blocks for nanoscale heterostructures, but little is understood about the physical principles governing their directed assembly. In-situ force microscopy was used to investigate organization of Cowpea Mosaic Virus engineered to bind specifically and reversibly at nanoscale chemical templates with sub-30nm features. Morphological evolution and assembly kinetics were measured as virus flux and inter-viral potential were varied. The resulting morphologies were similar to those of atomic-scale epitaxial systems, but the underlying thermodynamics was analogous to that of colloidal systems in confined geometries. The 1D templates biased the location of initial cluster formation, introduced asymmetric sticking probabilities, and drove 1D and 2D condensation at subcritical volume fractions. The growth kinetics followed a t{sup 1/2} law controlled by the slow diffusion of viruses. The lateral expansion of virus clusters that initially form on the 1D templates following introduction of polyethylene glycol (PEG) into the solution suggests a significant role for weak interaction.

  18. Direct Atom Imaging by Chemical-Sensitive Holography.

    PubMed

    Lühr, Tobias; Winkelmann, Aimo; Nolze, Gert; Krull, Dominique; Westphal, Carsten

    2016-05-11

    In order to understand the physical and chemical properties of advanced materials, functional molecular adsorbates, and protein structures, a detailed knowledge of the atomic arrangement is essential. Up to now, if subsurface structures are under investigation, only indirect methods revealed reliable results of the atoms' spatial arrangement. An alternative and direct method is three-dimensional imaging by means of holography. Holography was in fact proposed for electron waves, because of the electrons' short wavelength at easily accessible energies. Further, electron waves are ideal structure probes on an atomic length scale, because electrons have a high scattering probability even for light elements. However, holographic reconstructions of electron diffraction patterns have in the past contained severe image artifacts and were limited to at most a few tens of atoms. Here, we present a general reconstruction algorithm that leads to high-quality atomic images showing thousands of atoms. Additionally, we show that different elements can be identified by electron holography for the example of FeS2. PMID:27070050

  19. Unravelling Microbial Communities with DNA-Microarrays: Challengesand Future Directions.

    SciTech Connect

    Wagner, Michael; Smidt, Hauke; Loy, Alexander; Zhou, Jizhong

    2007-03-08

    High-throughput technologies are urgently needed formonitoring the formidable biodiversity and functional capabilities ofmicroorganisms in the environment. Ten years ago, DNA microarrays,miniaturized platforms for highly parallel hybridization reactions, foundtheir way into environmental microbiology and raised great expectationsamong researchers in the field. In this article, we briefly summarize thestate-of-the-art of microarray approaches in microbial ecology researchand discuss in more detail crucial problems and promising solutions.Finally, we outline scenarios for an innovative combination ofmicroarrays with other molecular tools for structure-function analysis ofcomplex microbial communities.

  20. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

    PubMed

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation. PMID:27001929

  1. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

    NASA Astrophysics Data System (ADS)

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-03-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

  2. Chemical Morphing of DNA Containing Four Noncanonical Bases.

    PubMed

    Eremeeva, Elena; Abramov, Michail; Margamuljana, Lia; Rozenski, Jef; Pezo, Valerie; Marlière, Philippe; Herdewijn, Piet

    2016-06-20

    The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo. PMID:27159019

  3. Chemical and Biological Consequences of Oxidatively Damaged Guanine in DNA

    PubMed Central

    Delaney, Sarah; Jarem, Daniel A.; Volle, Catherine B.; Yennie, Craig J.

    2013-01-01

    Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)·dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy·dGuo, and the hyperoxidized dGuo products. PMID:22239655

  4. Direct detection of linker DNA bending in defined-length oligomers of chromatin.

    PubMed Central

    Yao, J; Lowary, P T; Widom, J

    1990-01-01

    Linker DNA, which connects between nucleosomes in chromatin, is short and, therefore, may be essentially straight and inflexible. We have carried out hydrodynamic and electron microscopic studies of dinucleosomes--fragments of chromatin containing just two nucleosomes--to test the ability of linker DNA to bend. We find that ionic conditions that stabilize the folding of long chromatin cause linker DNA in dinucleosomes to bend, bringing the two nucleosomes into contact. The results uphold a key prediction of the solenoid model of chromosome folding and suggest a mechanism by which proteins that are separated along the DNA can interact by direct contact. Images PMID:2217191

  5. Dynamic and Progressive Control of DNA Origami Conformation by Modulating DNA Helicity with Chemical Adducts.

    PubMed

    Chen, Haorong; Zhang, Hanyu; Pan, Jing; Cha, Tae-Gon; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

    2016-05-24

    DNA origami has received enormous attention for its ability to program complex nanostructures with a few nanometer precision. Dynamic origami structures that change conformation in response to environmental cues or external signals hold great promises in sensing and actuation at the nanoscale. The reconfiguration mechanism of existing dynamic origami structures is mostly limited to single-stranded hinges and relies almost exclusively on DNA hybridization or strand displacement. Here, we show an alternative approach by demonstrating on-demand conformation changes with DNA-binding molecules, which intercalate between base pairs and unwind DNA double helices. The unwinding effect modulates the helicity mismatch in DNA origami, which significantly influences the internal stress and the global conformation of the origami structure. We demonstrate the switching of a polymerized origami nanoribbon between different twisting states and a well-constrained torsional deformation in a monomeric origami shaft. The structural transformation is shown to be reversible, and binding isotherms confirm the reconfiguration mechanism. This approach provides a rapid and reversible means to change DNA origami conformation, which can be used for dynamic and progressive control at the nanoscale. PMID:27057775

  6. A unified matrix hypothesis of DNA-directed morphogenesis, protodynamism and growth control.

    PubMed

    Scherrer, K

    1989-04-01

    A theoretical concept is proposed, in order to explain some enigmatic aspects of cellular and molecular biology of eukaryotic organisms. Among these are the C-value paradox of DNA redundancy, the correlation of DNA content and cell size, the disruption of genes at DNA level, the "Chromosome field" data of Lima de Faria (Hereditas 93:1, 1980), the "quantal mitosis" proposition of Holtzer et al. (Curr. Top. Dev. Biol. 7:229 1972), the inheritance of morphological patterns, the relations of DNA and chromosome organisation to cellular structure and function, the molecular basis of speciation, etc. The basic proposition of the "Unified Matrix Hypothesis" is that the nuclear DNA has a direct morphogenic function, in addition to its coding function in protein synthesis. This additional genetic information is thought to be largely contained in the non-protein coding transcribed DNA, and in the untranscribed part of the genome. PMID:2765661

  7. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    NASA Astrophysics Data System (ADS)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  8. 33 CFR 95.035 - Reasonable cause for directing a chemical test.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... chemical test. 95.035 Section 95.035 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND... DANGEROUS DRUG § 95.035 Reasonable cause for directing a chemical test. (a) Only a law enforcement officer or a marine employer may direct an individual operating a vessel to undergo a chemical test...

  9. Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays

    PubMed Central

    Reisner, Walter; Larsen, Niels B.; Flyvbjerg, Henrik; Tegenfeldt, Jonas O.; Kristensen, Anders

    2009-01-01

    We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into “connect-the-dots” conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA. PMID:19122138

  10. Incorporation of viral DNA packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA

    PubMed Central

    Haque, Farzin; Geng, Jia; Montemagno, Carlo; Guo, Peixuan

    2013-01-01

    Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression, and purification of the channel of bacteriophage phi29 DNA packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of dsDNA and chemicals. The membrane-embedded phi29 nanochannels remain functional and structurally intact under a range of conditions. When ions and macromolecules translocate through these nanochannels, reliable fingerprint changes in conductance are observed. Compared with other well studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis, and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around one month. PMID:23348364

  11. Development of a Daphnia magna DNA microarray for evaluating the toxicity of environmental chemicals.

    PubMed

    Watanabe, Hajime; Takahashi, Eri; Nakamura, Yuko; Oda, Shigeto; Tatarazako, Norihisa; Iguchi, Taisen

    2007-04-01

    Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism- and population-level responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna. Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide-based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or beta-naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical-specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism. PMID:17447551

  12. DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Ling; Yang, Chih-Feng; Agarwal, Vinay; Kim, Taehoon; Sonkusale, Sameer; Busnaina, Ahmed; Chen, Michelle; Dokmeci, Mehmet R.

    2010-03-01

    We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to ~ 300% and ~ 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications.

  13. DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry.

    PubMed

    Chen, Chia-Ling; Yang, Chih-Feng; Agarwal, Vinay; Kim, Taehoon; Sonkusale, Sameer; Busnaina, Ahmed; Chen, Michelle; Dokmeci, Mehmet R

    2010-03-01

    We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to approximately 300% and approximately 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications. PMID:20139486

  14. New directions: Atmospheric chemical mechanisms for the future

    EPA Science Inventory

    The chemical reaction scheme or mechanism used to represent atmospheric chemical reactions is at the heart of each air quality model used in research and policy applications to predict and analyse the complex air pollutants: ozone, air toxics and PM2.5. This is necessarily only a...

  15. A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.

    PubMed

    Yoo, S K; Yoon, M; Park, U J; Han, H S; Kim, J H; Hwang, H J

    1999-09-30

    High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA. PMID:10551259

  16. Direct Sequencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate

    PubMed Central

    Martínez-Priego, Llúcia; D’Auria, Giussepe; Calafell, Francesc; Moya, Andrés

    2014-01-01

    The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA. PMID:24887077

  17. New Modeling Approaches to Study DNA Damage by the Direct and Indirect Effects of Ionizing Radiation

    NASA Technical Reports Server (NTRS)

    Plante, Ianik; Cucinotta, Francis A.

    2012-01-01

    DNA is damaged both by the direct and indirect effects of radiation. In the direct effect, the DNA itself is ionized, whereas the indirect effect involves the radiolysis of the water molecules surrounding the DNA and the subsequent reaction of the DNA with radical products. While this problem has been studied for many years, many unknowns still exist. To study this problem, we have developed the computer code RITRACKS [1], which simulates the radiation track structure for heavy ions and electrons, calculating all energy deposition events and the coordinates of all species produced by the water radiolysis. In this work, we plan to simulate DNA damage by using the crystal structure of a nucleosome and calculations performed by RITRACKS. The energy deposition events are used to calculate the dose deposited in nanovolumes [2] and therefore can be used to simulate the direct effect of the radiation. Using the positions of the radiolytic species with a radiation chemistry code [3] it will be possible to simulate DNA damage by indirect effect. The simulation results can be compared with results from previous calculations such as the frequencies of simple and complex strand breaks [4] and with newer experimental data using surrogate markers of DNA double ]strand breaks such as . ]H2AX foci [5].

  18. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  19. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed Central

    Livneh, Z; Elad, D; Sperling, J

    1979-01-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  20. Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays.

    PubMed

    Lee, Chi-Ying; Harbers, Gregory M; Grainger, David W; Gamble, Lara J; Castner, David G

    2007-08-01

    Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity. PMID:17625851

  1. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

    PubMed Central

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation. PMID:27001929

  2. A Pol V–Mediated Silencing, Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA

    PubMed Central

    Douet, Julien; Tutois, Sylvie; Tourmente, Sylvette

    2009-01-01

    The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA–directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA–Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA–encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V–loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM–independent and comes specifically at chromosome 4, in addition to the RdDM pathway. PMID:19834541

  3. Invited Review: DNA-mediated artificial nanobiostructures: state of the art and future directions

    NASA Astrophysics Data System (ADS)

    Bashir, R.

    2001-01-01

    DNA (deoxyribonucleic acid)-mediated assembly of nanometer and micrometer scale structures can have a profound impact in the fields of nanoelectronics and nanotechnology. Such structures can also find applications in microelectromechanical systems, hybrid bio-sensors, and the potential to continue the scaling of Moore's law beyond the 50 nm node. While engineers and scientists have been long aspiring to controllably and specifically manipulate structures at the micrometer and nanometer scale, nature has been performing these tasks and assembling structures with great accuracy and high efficiency using highly specific biological molecules such as DNA and proteins. This paper describes the motivations and fundamentals behind these assembly concepts, with a focus on DNA hybridization-mediated assembly, and presents the state of the art in this field. In addition, new ideas and directions for future research on DNA-mediated assembly of active devices and DNA-based molecular devices are also presented.

  4. A comparison of RNA with DNA in template-directed synthesis

    NASA Technical Reports Server (NTRS)

    Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

  5. Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

    PubMed Central

    Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

  6. Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures.

    PubMed

    Miller, Diana T; Grimwade, Julia E; Betteridge, Thu; Rozgaja, Tania; Torgue, Julien J-C; Leonard, Alan C

    2009-11-01

    Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly. PMID:19833870

  7. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    PubMed Central

    2011-01-01

    Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

  8. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders?

    PubMed Central

    Keil, Kimberly P.; Lein, Pamela J.

    2016-01-01

    There is now compelling evidence that gene by environment interactions are important in the etiology of autism spectrum disorders (ASDs). However, the mechanisms by which environmental factors interact with genetic susceptibilities to confer individual risk for ASD remain a significant knowledge gap in the field. The epigenome, and in particular DNA methylation, is a critical gene expression regulatory mechanism in normal and pathogenic brain development. DNA methylation can be influenced by environmental factors such as diet, hormones, stress, drugs, or exposure to environmental chemicals, suggesting that environmental factors may contribute to adverse neurodevelopmental outcomes of relevance to ASD via effects on DNA methylation in the developing brain. In this review, we describe epidemiological and experimental evidence implicating altered DNA methylation as a potential mechanism by which environmental chemicals confer risk for ASD, using polychlorinated biphenyls (PCBs), lead, and bisphenol A (BPA) as examples. Understanding how environmental chemical exposures influence DNA methylation and how these epigenetic changes modulate the risk and/or severity of ASD will not only provide mechanistic insight regarding gene-environment interactions of relevance to ASD but may also suggest potential intervention strategies for these and potentially other neurodevelopmental disorders. PMID:27158529

  9. Direct electrochemical stripping detection of cystic-fibrosis-related DNA linked through cadmium sulfide quantum dots

    NASA Astrophysics Data System (ADS)

    Marin, Sergio; Merkoçi, Arben

    2009-02-01

    Electrochemical detection of a cadmium sulfide quantum dots (CdS QDs)-DNA complex connected to paramagnetic microbeads (MB) was performed without the need for chemical dissolving. The method is based on dropping 20 µl of CdS QD-DNA-MB suspension on the surface of a screen-printed electrode. It is followed by magnetic collection on the surface of the working electrode and electrochemical detection using square-wave voltammetry (SWV), giving a well-shaped and sensitive analytical signal. A cystic-fibrosis-related DNA sequence was sandwiched between the two DNA probes. One DNA probe is linked via biotin-streptavidin bonding with MB and the other one via thiol groups with the CdS QD used as tags. Nonspecific signals of DNA were minimized using a blocking agent and the results obtained were successfully employed in a model DNA sensor with an interest in future applications in the clinical field. The developed nanoparticle biosensing system may offer numerous opportunities in other fields where fast, low cost and efficient detection of small volume samples is required.

  10. Direct Correlation of DNA Binding and Single Protein Domain Motion via Dual Illumination Fluorescence Microscopy

    PubMed Central

    2015-01-01

    We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. PMID:25204359

  11. Direct non transcriptional role of NF-Y in DNA replication.

    PubMed

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. PMID:26732297

  12. Having a direct look: Analysis of DNA damage and repair mechanisms by next generation sequencing

    PubMed Central

    Meier, Bettina; Gartner, Anton

    2014-01-01

    Genetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis. PMID:25131498

  13. Mismatch repair inhibits homeologous recombination via coordinated directional unwinding of trapped DNA structures.

    PubMed

    Tham, Khek-Chian; Hermans, Nicolaas; Winterwerp, Herrie H K; Cox, Michael M; Wyman, Claire; Kanaar, Roland; Lebbink, Joyce H G

    2013-08-01

    Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability. PMID:23932715

  14. Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

    PubMed Central

    Maeda, Naoyoshi; Palmarini, Massimo; Murgia, Claudio; Fan, Hung

    2001-01-01

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2–3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential. PMID:11296288

  15. Rapid DNA Sequencing by Direct Nanoscale Reading of Nucleotide Bases on Individual DNA Chains

    SciTech Connect

    Lee, James Weifu; Meller, Amit

    2007-01-01

    Since the independent invention of DNA sequencing by Sanger and by Gilbert 30 years ago, it has grown from a small scale technique capable of reading several kilobase-pair of sequence per day into today's multibillion dollar industry. This growth has spurred the development of new sequencing technologies that do not involve either electrophoresis or Sanger sequencing chemistries. Sequencing by Synthesis (SBS) involves multiple parallel micro-sequencing addition events occurring on a surface, where data from each round is detected by imaging. New High Throughput Technologies for DNA Sequencing and Genomics is the second volume in the Perspectives in Bioanalysis series, which looks at the electroanalytical chemistry of nucleic acids and proteins, development of electrochemical sensors and their application in biomedicine and in the new fields of genomics and proteomics. The authors have expertly formatted the information for a wide variety of readers, including new developments that will inspire students and young scientists to create new tools for science and medicine in the 21st century. Reviews of complementary developments in Sanger and SBS sequencing chemistries, capillary electrophoresis and microdevice integration, MS sequencing and applications set the framework for the book.

  16. New directions: Atmospheric chemical mechanisms for the future

    NASA Astrophysics Data System (ADS)

    Kaduwela, Ajith; Luecken, Deborah; Carter, William; Derwent, Richard

    2015-12-01

    The atmospheric chemical reaction scheme, commonly referred to as the chemical mechanism, is the portion of an air quality model that represents the atmospheric chemistry of the pollutants. It is at the heart of every air quality model used in research and policy applications to predict and analyse the complex air pollutants: ozone, air toxics and PM2.5. The chemical mechanism should incorporate available information on chemical kinetics and reaction pathways and be the conduit through which the fundamental science of atmospheric chemistry is applied to solve real-world problems. The efficiency and effectiveness of policies developed to reduce exposure to harmful pollutants depend on how well the mechanisms reflect the actual chemistry. If the mechanism has reaction pathways that are incorrectly characterised or completely missing, the resulting predictions may underestimate emission reduction requirements needed to meet public health and ecosystem protection targets, or may overstate the emission reductions needed and cause unnecessary implementation costs. It is therefore essential that mechanisms utilise the best, most up-to-date atmospheric chemistry information available so that policy development is based on air quality model predictions that are robust, transparent and free from scientific challenge. We are concerned that this may not continue to be the case.

  17. Chemical Reaction Engineering: Current Status and Future Directions.

    ERIC Educational Resources Information Center

    Dudukovic, M. P.

    1987-01-01

    Describes Chemical Reaction Engineering (CRE) as the discipline that quantifies the interplay of transport phenomena and kinetics in relating reactor performance to operating conditions and input variables. Addresses the current status of CRE in both academic and industrial settings and outlines future trends. (TW)

  18. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology.

    PubMed

    Cheung-Ong, Kahlin; Giaever, Guri; Nislow, Corey

    2013-05-23

    DNA-damaging agents have a long history of use in cancer chemotherapy. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. Novel anticancer compounds are continually being developed in the hopes of addressing these limitations; however, it is essential to be able to evaluate these compounds for their mechanisms of action. This review covers the current DNA-damaging agents used in the clinic, discusses their limitations, and describes the use of chemical genomics to uncover new information about the DNA damage response network and to evaluate novel DNA-damaging compounds. PMID:23706631

  19. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    PubMed

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  20. DNA-decorated carbon nanotube-based FETs as ultrasensitive chemical sensors: Discrimination of homologues, structural isomers, and optical isomers

    NASA Astrophysics Data System (ADS)

    Khamis, S. M.; Jones, R. A.; Johnson, A. T. C.; Preti, G.; Kwak, J.; Gelperin, A.

    2012-06-01

    We have explored the abilities of all-electronic DNA-carbon nanotube (DNA-NT) vapor sensors to discriminate very similar classes of molecules. We screened hundreds of DNA-NT devices against a panel of compounds chosen because of their similarities. We demonstrated that DNA-NT vapor sensors readily discriminate between series of chemical homologues that differ by single methyl groups. DNA-NT devices also discriminate among structural isomers and optical isomers, a trait common in biological olfactory systems, but only recently demonstrated for electronic FET based chemical sensors.

  1. Suppression of the chemically transformed phenotype of BHK cells by a human cDNA

    SciTech Connect

    Eiden, M.V.; MacArthur, L.; Okayama, Hiroto )

    1991-10-01

    Transformation of baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis. A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

  2. Understanding of DNA directed nanoparticle superlattices in bulk and thin film

    NASA Astrophysics Data System (ADS)

    Lee, Byeongdu

    Over the years, there have been significant advances in assembling nanoparticles with DNA into superlattices. Since the first reports on DNA directed FCC and BCC superlattices consisting of single type of spherical nanoparticles, building blocks for the DNA-nanoparticle superlattices have been extended from a spherical gold nanoparticle to various types of other particles including quantum dots, magnetic, hollow, or polyhedral particles. Not only single component, but superlattices of binary and ternary components have also been synthesized. Although still many details are unclear, now there is a general consensus about thermodynamics of this type of assembly, which led us to fabricate thin films of DNA directed nanoparticle superlattices on substrate for applications such as optical materials. Since the structures are formed in aqueous condition, small angle x-ray scattering (SAXS) that does not disturb the system has been a critical tool to determine structural and thermodynamic characteristics of the assemblies. Thus, we have also been improving SAXS instrumentations and computational methods to calculate scattering profiles for the nanoparticle superlattices. In this talk, we will summarize our works with a focus on some structural details of these superlattices and DNA and understanding about the role of DNA in the crystallization processes in bulk and thin film.

  3. A Binary-Encounter-Bethe Approach to Simulate DNA Damage by the Direct Effect

    NASA Technical Reports Server (NTRS)

    Plante, Ianik; Cucinotta, Francis A.

    2013-01-01

    The DNA damage is of crucial importance in the understanding of the effects of ionizing radiation. The main mechanisms of DNA damage are by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research in this area, many questions on the formation of DNA damage remains. To refine existing DNA damage models, an approach based on the Binary-Encounter-Bethe (BEB) model was developed[1]. This model calculates differential cross sections for ionization of the molecular orbitals of the DNA bases, sugars and phosphates using the electron binding energy, the mean kinetic energy and the occupancy number of the orbital. This cross section has an analytic form which is quite convenient to use and allows the sampling of the energy loss occurring during an ionization event. To simulate the radiation track structure, the code RITRACKS developed at the NASA Johnson Space Center is used[2]. This code calculates all the energy deposition events and the formation of the radiolytic species by the ion and the secondary electrons as well. We have also developed a technique to use the integrated BEB cross section for the bases, sugar and phosphates in the radiation transport code RITRACKS. These techniques should allow the simulation of DNA damage by ionizing radiation, and understanding of the formation of double-strand breaks caused by clustered damage in different conditions.

  4. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

  5. Fluorescent marker for direct detection of specific dsDNA sequences

    PubMed Central

    Dylla-Spears, Rebecca; Townsend, Jacqueline E.; Sohn, Lydia L.; Jen-Jacobson, Linda; Muller, Susan J.

    2009-01-01

    We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme’s binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker’s ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods. PMID:19908852

  6. Fluorescent marker for direct detection of specific dsDNA sequences.

    PubMed

    Dylla-Spears, Rebecca; Townsend, Jacqueline E; Sohn, Lydia L; Jen-Jacobson, Linda; Muller, Susan J

    2009-12-15

    We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods. PMID:19908852

  7. Direct chemical synthesis of L10 FePt nanoparticles

    NASA Astrophysics Data System (ADS)

    Tzitzios, Vasilis; Basina, Georgia; Colak, Levent; Niarchos, Dimitrios; Hadjipanayis, George

    2011-04-01

    Ordered faced-centered tetragonal (fct) FePt nanoparticles were successfully synthesized by a chemical method using presynthesized Au nanoparticles as the "catalyst." The reaction temperature was also studied and it seems that there is an optimum value of 360 °C where the fct structure is formed. The particles have a mean diameter of 3.5-15 nm and the x-ray diffraction patterns exhibited (001) and (110), which signified the tetragonal phase formation. Room temperature magnetic hysteresis loops show that the FePt particles have coercive fields between 0.68 and 2.8 kOe.

  8. Direct action of endocrine disrupting chemicals on human sperm.

    PubMed

    Schiffer, Christian; Müller, Astrid; Egeberg, Dorte L; Alvarez, Luis; Brenker, Christoph; Rehfeld, Anders; Frederiksen, Hanne; Wäschle, Benjamin; Kaupp, U Benjamin; Balbach, Melanie; Wachten, Dagmar; Skakkebaek, Niels E; Almstrup, Kristian; Strünker, Timo

    2014-07-01

    Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca(2+) increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca(2+) levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. PMID:24820036

  9. Direct action of endocrine disrupting chemicals on human sperm

    PubMed Central

    Schiffer, Christian; Müller, Astrid; Egeberg, Dorte L; Alvarez, Luis; Brenker, Christoph; Rehfeld, Anders; Frederiksen, Hanne; Wäschle, Benjamin; Kaupp, U Benjamin; Balbach, Melanie; Wachten, Dagmar; Skakkebaek, Niels E; Almstrup, Kristian; Strünker, Timo

    2014-01-01

    Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. PMID:24820036

  10. Direct-Dispense Polymeric Waveguides Platform for Optical Chemical Sensors

    PubMed Central

    Hajj-Hassan, Mohamad; Gonzalez, Timothy; Ghafar-Zadeh, Ebrahim; Djeghelian, Hagop; Chodavarapu, Vamsy; Andrews, Mark; Therriault, Daniel

    2008-01-01

    We describe an automated robotic technique called direct-dispense to fabricate a polymeric platform that supports optical sensor arrays. Direct-dispense, which is a type of the emerging direct-write microfabrication techniques, uses fugitive organic inks in combination with cross-linkable polymers to create microfluidic channels and other microstructures. Specifically, we describe an application of direct-dispensing to develop optical biochemical sensors by fabricating planar ridge waveguides that support sol-gel-derived xerogel-based thin films. The xerogel-based sensor materials act as host media to house luminophore biochemical recognition elements. As a prototype implementation, we demonstrate gaseous oxygen (O2) responsive optical sensors that operate on the basis of monitoring luminescence intensity signals. The optical sensor employs a Light Emitting Diode (LED) excitation source and a standard silicon photodiode as the detector. The sensor operates over the full scale (0%-100%) of O2 concentrations with a response time of less than 1 second. This work has implications for the development of miniaturized multi-sensor platforms that can be cost-effectively and reliably mass-produced.

  11. Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

    NASA Astrophysics Data System (ADS)

    Swasey, Steven M.; Gwinn, Elisabeth G.

    2016-04-01

    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 Mg2+, 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’.

  12. DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

    PubMed

    Seymour, Elif; Daaboul, George G; Zhang, Xirui; Scherr, Steven M; Ünlü, Nese Lortlar; Connor, John H; Ünlü, M Selim

    2015-10-20

    Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications. PMID:26378807

  13. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/. PMID:24847672

  14. Dependence of 5-fluorouracil-mediated radiosensitization on DNA-directed effects

    SciTech Connect

    Lawrence, T.S.; Davis, M.A.; Maybaum, J. )

    1994-06-15

    Although 5-fluorouracil (FUra) has been demonstrated to be a radiation sensitizer both in the laboratory and the clinic, it is not known whether radiosensitization results primary from FUra's DNA or RNA-directed effects. The authors studied the radiosensitizing effects of FUra [+-] thymidine (dThd) on HT29 human colon cancer cells, which are relatively sensitive to the DNA-directed action of FUra, in comparison to SW620 and HuTu80 human colon cancer cells, which are relatively resistant to FUra's DNA-directed effects. They hypothesized that if FUra were acting chiefly through DNA dependent mechanisms, HT29 cells would (a) show greater radiosensitization than SW620 and HuTu80 cells under the same conditions of exposure; and (b) demonstrate selective reversal of radiation sensitivity (compared to cytotoxicity) in the presence of FUra + dThd, compared to FUra alone. They found that the enhancement ratio produced by a 24 h exposure to 10 [mu]M FUra was significantly greater in HT29 cells compared to SW620 and HuTu80 cells (enhancement ratios of 2.1 [+-] 0.1; 1.1 [+-] 0.1, and 1.3 [+-] 0.1, respectively). Furthermore, in HT29 cells, dThd blocked FUra-mediated radiosensitization to a greater extent than FUra-mediated cytotoxicity. Thus, the hypotheses were confirmed. These findings support the concept that the manipulation of FUra's DNA-dependent actions, for example, through modulators of thymidylate synthase (TS) activity, may increase radiosensitization in clinical trials in the treatment of gastrointestinal cancers. However, since resistance to the DNA-directed effects of fluoropyrimidines can result from mechanisms unrelated to TS inhibition, additional strategies will be required to potentiate fluoropyrimidine-mediated radiosensitization. 15 refs., 2 figs., 1 tab.

  15. Macroscopic response and directional disorder dynamics in chemically substituted ferroelectrics

    NASA Astrophysics Data System (ADS)

    Parravicini, Jacopo; DelRe, Eugenio; Agranat, Aharon J.; Parravicini, Gianbattista

    2016-03-01

    Using temperature-resolved dielectric spectroscopy in the range 25-320 K we investigate the macroscopic response, phase symmetry, and order/disorder states in bulk ferroelectric K1-yLiyTa1-xNbx (KLTN). Four long-range symmetry phases are identified with their relative transitions. Directional analysis of the order/disorder states using Fröhlich entropy indicates global symmetry breaking along the growth axis and an anisotropic dipolar effective thermodynamic behavior, which ranges from disordered to ordered at the same temperature for different directions in the sample. Results indicate that the macroscopic polarization, driven by nanosized polar regions, follows a microscopic perovskite eight-sites lattice model.

  16. DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.

    PubMed

    Costa, M; Zhitkovich, A; Harris, M; Paustenbach, D; Gargas, M

    1997-04-11

    Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that

  17. Direct evaluation of airborne contamination in chemically amplified resist films

    NASA Astrophysics Data System (ADS)

    Yamashita, Yoshio; Taguchi, Takao; Watanabe, Takeo

    1995-06-01

    Airborne contamination in chemically amplified resist films was evaluated by monitoring deprotection reaction using an IR spectrometer. T-BOC protected (20, 50 and 100 mol%) m- and p-cresol novolak resins and triphenyltriflate were used as a matrix polymer and a photoacid generator (PAG), respectively. Three levels of clean environments whose base contaminant (NH4+) concentrations were 50 - 80, 5 - 10 and less than 1 ppb, were prepared for the experiments. In order to determine the delay effects precisely, other processes including baking, exposure, and storage during process intervals were conducted in a base-free environment. The PEB delay effect as well as radiation sensitivity without delay depended on the t-BOC content, and the best results were obtained at 50% and 25 - 50% t- BOC contents in m-cresol novolak and p-cresol novolak systems, respectively.

  18. Environmental chemicals and DNA methylation in adults: a systematic review of the epidemiologic evidence.

    PubMed

    Ruiz-Hernandez, Adrian; Kuo, Chin-Chi; Rentero-Garrido, Pilar; Tang, Wan-Yee; Redon, Josep; Ordovas, Jose M; Navas-Acien, Ana; Tellez-Plaza, Maria

    2015-01-01

    Current evidence supports the notion that environmental exposures are associated with DNA-methylation and expression changes that can impact human health. Our objective was to conduct a systematic review of epidemiologic studies evaluating the association between environmental chemicals with DNA methylation levels in adults. After excluding arsenic, recently evaluated in a systematic review, we identified a total of 17 articles (6 on cadmium, 4 on lead, 2 on mercury, 1 on nickel, 1 on antimony, 1 on tungsten, 5 on persistent organic pollutants and perfluorinated compounds, 1 on bisphenol A, and 3 on polycyclic aromatic hydrocarbons). The selected articles reported quantitative methods to determine DNA methylation including immunocolorimetric assays for total content of genomic DNA methylation, and microarray technologies, methylation-specific quantitative PCR, Luminometric Methylation Assay (LUMA), and bisulfite pyrosequencing for DNA methylation content of genomic sites such as gene promoters, LINE-1, Alu elements, and others. Considering consistency, temporality, strength, dose-response relationship, and biological plausibility, we concluded that the current evidence is not sufficient to provide inference because differences across studies and limited samples sizes make it difficult to compare across studies and to evaluate sources of heterogeneity. Important questions for future research include the need for larger and longitudinal studies, the validation of findings, and the systematic evaluation of the dose-response relationships. Future studies should also consider the evaluation of epigenetic marks recently in the research spotlight such as DNA hydroxymethylation and the role of underlying genetic variants. PMID:25984247

  19. Direct numerical simulation of turbulent, chemically reacting flows

    NASA Astrophysics Data System (ADS)

    Doom, Jeffrey Joseph

    This dissertation: (i) develops a novel numerical method for DNS/LES of compressible, turbulent reacting flows, (ii) performs several validation simulations, (iii) studies auto-ignition of a hydrogen vortex ring in air and (iv) studies a hydrogen/air turbulent diffusion flame. The numerical method is spatially non-dissipative, implicit and applicable over a range of Mach numbers. The compressible Navier-Stokes equations are rescaled so that the zero Mach number equations are discretely recovered in the limit of zero Mach number. The dependent variables are co--located in space, and thermodynamic variables are staggered from velocity in time. The algorithm discretely conserves kinetic energy in the incompressible, inviscid, non--reacting limit. The chemical source terms are implicit in time to allow for stiff chemical mechanisms. The algorithm is readily applicable to complex chemical mechanisms. Good results are obtained for validation simulations. The algorithm is used to study auto-ignition in laminar vortex rings. A nine species, nineteen reaction mechanism for H2/air combustion proposed by Mueller et al. [37] is used. Diluted H 2 at ambient temperature (300 K) is injected into hot air. The simulations study the effect of fuel/air ratio, oxidizer temperature, Lewis number and stroke ratio (ratio of piston stroke length to diameter). Results show that auto--ignition occurs in fuel lean, high temperature regions with low scalar dissipation at a 'most reactive' mixture fraction, zeta MR (Mastorakos et al. [32]). Subsequent evolution of the flame is not predicted by zetaMR; a most reactive temperature TMR is defined and shown to predict both the initial auto-ignition as well as subsequent evolution. For stroke ratios less than the formation number, ignition in general occurs behind the vortex ring and propagates into the core. At higher oxidizer temperatures, ignition is almost instantaneous and occurs along the entire interface between fuel and oxidizer. For stroke

  20. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

    SciTech Connect

    Ausin, Israel; Greenberg, Maxim V.C.; Simanshu, Dhirendra K.; Hale, Christopher J.; Vashisht, Ajay A.; Simon, Stacey A.; Lee, Tzuu-fen; Feng, Suhua; Española, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2012-10-23

    At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

  1. Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

    SciTech Connect

    Karger, A.E.; Weiss, R.; Gesteland, R.F. Eccles Inst. of Human Genetics, Salt Lake City, UT )

    1993-07-01

    A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.

  2. Directly observing the motion of DNA molecules near solid-state nanopores.

    PubMed

    Ando, Genki; Hyun, Changbae; Li, Jiali; Mitsui, Toshiyuki

    2012-11-27

    We investigate the diffusion and the drift motion of λ DNA molecules near solid-state nanopores prior to their translocation through the nanopores using fluorescence microscopy. The radial dependence of the electric field near a nanopore generated by an applied voltage in ionic solution can be estimated quantitatively in 3D by analyzing the motion of negatively charged DNA molecules. We find that the electric field is approximately spherically symmetric around the nanopore under the conditions investigated. In addition, DNA clogging at the nanopore was directly observed. Surprisingly, the probability of the clogging event increases with increasing external bias voltage. We also find that DNA molecules clogging the nanopore reduce the electric field amplitude at the nanopore membrane surface. To better understand these experimental results, analytical method with Ohm's law and computer simulation with Poisson and Nernst-Planck (PNP) equations are used to calculate the electric field near the nanopore. These results are of great interest in both experimental and theoretical considerations of the motion of DNA molecules near voltage-biased nanopores. These findings will also contribute to the development of solid-state nanopore-based DNA sensing devices. PMID:23046052

  3. Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses

    PubMed Central

    Kichaev, Gleb; Mendoza, Janess M; Amante, Dinah; Smith, Trevor RF; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2013-01-01

    In vivo electroporation (EP) has been shown to be a highly efficient non-viral method for enhancing DNA vaccine delivery and immunogenicity, when the site of immunization is the skin or muscle of animals and humans. However, the route of entry for many microbial pathogens is via the mucosal surfaces of the human body. We have previously reported on minimally invasive, surface and contactless EP devices for enhanced DNA delivery to dermal tissue. Robust antibody responses were induced following vaccine delivery in several tested animal models using these devices. Here, we investigated extending the modality of the surface device to efficiently deliver DNA vaccines to mucosal tissue. Initially, we demonstrated reporter gene expression in the epithelial layer of buccal mucosa in a guinea pig model. There was minimal tissue damage in guinea pig mucosal tissue resulting from EP. Delivery of a DNA vaccine encoding influenza virus nucleoprotein (NP) of influenza H1N1 elicited robust and sustained systemic IgG antibody responses following EP-enhanced delivery in the mucosa. Upon further analysis, IgA antibody responses were detected in vaginal washes and sustained cellular immune responses were detected in animals immunized at the oral mucosa with the surface EP device. This data confirms that DNA delivery and EP targeting mucosal tissue directly results in both robust and sustainable humoral as well as cellular immune responses without tissue damage. These responses are seen both in the mucosa and systemically in the blood. Direct DNA vaccine delivery enhanced by EP in mucosa may have important clinical applications for delivery of prophylactic and therapeutic DNA vaccines against diseases such as HIV, HPV and pneumonia that enter at mucosal sites and require both cellular and humoral immune responses for protection. PMID:23954979

  4. Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses.

    PubMed

    Kichaev, Gleb; Mendoza, Janess M; Amante, Dinah; Smith, Trevor R F; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2013-10-01

    In vivo electroporation (EP) has been shown to be a highly efficient non-viral method for enhancing DNA vaccine delivery and immunogenicity, when the site of immunization is the skin or muscle of animals and humans. However, the route of entry for many microbial pathogens is via the mucosal surfaces of the human body. We have previously reported on minimally invasive, surface and contactless EP devices for enhanced DNA delivery to dermal tissue. Robust antibody responses were induced following vaccine delivery in several tested animal models using these devices. Here, we investigated extending the modality of the surface device to efficiently deliver DNA vaccines to mucosal tissue. Initially, we demonstrated reporter gene expression in the epithelial layer of buccal mucosa in a guinea pig model. There was minimal tissue damage in guinea pig mucosal tissue resulting from EP. Delivery of a DNA vaccine encoding influenza virus nucleoprotein (NP) of influenza H1N1 elicited robust and sustained systemic IgG antibody responses following EP-enhanced delivery in the mucosa. Upon further analysis, IgA antibody responses were detected in vaginal washes and sustained cellular immune responses were detected in animals immunized at the oral mucosa with the surface EP device. This data confirms that DNA delivery and EP targeting mucosal tissue directly results in both robust and sustainable humoral as well as cellular immune responses without tissue damage. These responses are seen both in the mucosa and systemically in the blood. Direct DNA vaccine delivery enhanced by EP in mucosa may have important clinical applications for delivery of prophylactic and therapeutic DNA vaccines against diseases such as HIV, HPV and pneumonia that enter at mucosal sites and require both cellular and humoral immune responses for protection. PMID:23954979

  5. 33 CFR 95.035 - Reasonable cause for directing a chemical test.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Reasonable cause for directing a... DANGEROUS DRUG § 95.035 Reasonable cause for directing a chemical test. (a) Only a law enforcement officer... reasonable cause exists. Reasonable cause exists when: (1) The individual was directly involved in...

  6. The Chemical Synthesis of DNA/RNA: Our Gift to Science

    PubMed Central

    Caruthers, Marvin H.

    2013-01-01

    It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs. PMID:23223445

  7. Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

    PubMed Central

    Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

    2013-01-01

    Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

  8. Direct amplification of DNA from fresh and preserved ectomycorrhizal root tips.

    PubMed

    Bent, Elizabeth; Taylor, D Lee

    2010-02-01

    Methods are described by which DNA can be amplified directly from ectomycorrhizal root tip homogenates of a variety of plant species (Picea mariana (black spruce), Betula papyrifera (paper birch), Populus tremuloides (trembling aspen) and Alnus sp.(alder)), including root tips that have been preserved in RNA Later (Ambion, Austin, TX). In most cases for extracts and homogenates diluted 10-fold prior to PCR, and in all cases for 100-fold dilutions, direct amplification of DNA from fresh root tip homogenates yielded as many or more ng of PCR amplicon (fungal ITS region) than amplification of DNA extracted from the same tips using a commercial kit or a manual ethanol precipitation-based method. For alder root tip extracts diluted 10-fold, the commercial kit method yielded more ng of PCR amplicon than 10-fold diluted, although direct use of homogenates still resulted in amplification in all tips tested. We also demonstrate consistent amplification of DNA from homogenates of birch, spruce and aspen ectomycorrhizal root tips preserved for 4months in RNA Later. PMID:19963016

  9. Chemical reactions occurring during direct solar reduction of CO2.

    PubMed

    Lyma, J L; Jensen, R J

    2001-09-28

    At high temperatures carbon dioxide may absorb solar radiation and react to form carbon monoxide and molecular oxygen. The CO, so produced, may be converted by well-established means to a combustible fuel, such as methanol. We intend to make a future demonstration of the solar reduction of CO2 based on these processes. This paper, however, addresses only the problem of preserving, or even enhancing, the initial photolytic CO by quenching the hot gas with colder H2O or CO2. We present model calculations with a reaction mechanism used extensively in other calculations. If a CO2 gas stream is heated and photolyzed by intense solar radiation and then allowed to cool slowly, it will react back to the initial CO2 by a series of elementary chemical reactions. The back reaction to CO2 can be terminated with the rapid addition of CO2, water, or a mixture. Calculations show that a three-fold quench with pure CO2 will stop the reactions and preserve over 90% of the initial photolytic CO. We find that water has one of two effects. It can either increase the CO level, or it can catalyze the recombination of O and CO to CO2. The gas temperature is the determining factor. If the quench gas is not sufficient to keep the temperature below approximately 1100 K, a chain-branching reaction dominates and the reaction to CO2 occurs. If the temperature stays below that level a chain terminating reaction dominates and the CO is increased. The former case occurs below approximately a fourfold quench with a water/CO2 mixture. The later case occurs when the quench is greater than fourfold. We conclude that CO2, H2O, or a mixture may quench the hot gas stream photolyzed by solar radiation and preserve the photolytic CO. PMID:11589409

  10. A direct detection of Escherichia coli genomic DNA using gold nanoprobes

    PubMed Central

    2012-01-01

    Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without

  11. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

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

    PubMed

    He, Gen; Li, Jie; Ci, Haina; Qi, Chuanmin; Guo, Xuefeng

    2016-07-25

    Herein, we report label-free detection of single-molecule DNA hybridization dynamics with single-base resolution. By using an electronic circuit based on point-decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal-to-noise ratio and bandwidth. These measurements reveal two-level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base-by-base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base-pair level. This measurement capability promises a label-free single-molecule approach to probe biomolecular interactions with fast dynamics. PMID:27272178

  13. Brownian dynamics simulation of the nucleosome chirality - the wrapping direction of DNA on the histone octamer

    NASA Astrophysics Data System (ADS)

    Wang, Peng-Ye

    2005-03-01

    In eukaryote nucleosome, DNA wraps around a histone octamer in a left-handed way. We study the process of chirality formation of nucleosome with Brownian dynamics simulation. We model the histone octamer with a quantitatively adjustable chirality: left-handed, right-handed or non-chiral, and simulate the dynamical wrapping process of a DNA molecule on it. We find that the chirality of a nucleosome formed is strongly dependent on that of the histone octamer, and different chiralities of the histone octamer induce its different rotation directions in the wrapping process of DNA. In addition, a very weak chirality of the histone octamer is quite enough for sustaining the correct chirality of the nucleosome formed. We also show that the chirality of a nucleosome may be broken at elevated temperature.

  14. DNA hybridization as a guide to phylogeny: chemical and physical limits.

    PubMed

    Schmid, C W; Marks, J

    1990-03-01

    The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance. Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments. As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes. PMID:2109086

  15. DIRECT-ACTING, DNA-DAMAGING AS (III)-METHYLATED SPECIES: IMPLICATIONS FOR A CARCINOGENIC MECHANISM OF ACTION OF ARSENICALS

    EPA Science Inventory

    Direct-acting, DNA-damaging As (III)-methylated species: implications for a carcinogenic . mechanism of action of arsenicals

    Inorganic arsenic (iAs, arsenite and arsenate) has been thought to act as a carcinogen without reacting directly with DNA; neither iAs nor the As(...

  16. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    SciTech Connect

    Miller, M.R.; Heyneman, C.; Walker, S.; Ulrich, R.G.

    1986-03-01

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S/sub 1/ nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by (/sup 3/H)TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.

  17. One-Directional Fluidic Flow Induced by Chemical Wave Propagation in a Microchannel.

    PubMed

    Arai, Miyu; Takahashi, Kazuhiro; Hattori, Mika; Hasegawa, Takahiko; Sato, Mami; Unoura, Kei; Nabika, Hideki

    2016-05-26

    A one-directional flow induced by chemical wave propagation was investigated to understand the origin of its dynamic flow. A cylindrical injection port was connected with a straight propagation channel; the chemical wave was initiated at the injection port. Chemical waves propagated with a constant velocity irrespective of the channel width, indicating that the dynamics of the chemical waves were governed by a geometry-independent interplay between the chemical reaction and diffusion. In contrast, the velocity of the one-directional flow was dependent on the channel width. Furthermore, enlargement of the injection port volume increased the flow velocity and volume flux. These results imply that the one-directional flow in the microchannel is due to a hydrodynamic effect induced in the injection port. Spectroscopic analysis of a pH indicator revealed the simultaneous behavior between the pH increase near the injection port and the one-directional flow. Hence, we can conclude that the one-directional flow in the microchannel with chemical wave propagation was caused by a proton consumption reaction in the injection port, probably through liquid volume expansion by the reaction products and the reaction heat. It is a characteristic feature of the present system that the hydrodynamic flow started from the chemical wave initiation point and not the propagation wavefront, as observed for previous systems. PMID:27167307

  18. Specific and reversible DNA-directed self-assembly of oil-in-water emulsion droplets

    PubMed Central

    Hadorn, Maik; Boenzli, Eva; Sørensen, Kristian T.; Fellermann, Harold; Eggenberger Hotz, Peter; Hanczyc, Martin M.

    2012-01-01

    Higher-order structures that originate from the specific and reversible DNA-directed self-assembly of microscopic building blocks hold great promise for future technologies. Here, we functionalized biotinylated soft colloid oil-in-water emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker. We show the components of this modular linking system to be stable and to induce sequence-specific aggregation of binary mixtures of emulsion droplets. Three length scales were thereby involved: nanoscale DNA base pairing linking microscopic building blocks resulted in macroscopic aggregates visible to the naked eye. The aggregation process was reversible by changing the temperature and electrolyte concentration and by the addition of competing oligonucleotides. The system was reset and reused by subsequent refunctionalization of the emulsion droplets. DNA-directed self-assembly of oil-in-water emulsion droplets, therefore, offers a solid basis for programmable and recyclable soft materials that undergo structural rearrangements on demand and that range in application from information technology to medicine. PMID:23175791

  19. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    NASA Astrophysics Data System (ADS)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  20. Direct detection of DNA methylation during single-molecule, real-time sequencing

    PubMed Central

    Flusberg, Benjamin A.; Webster, Dale; Lee, Jessa; Travers, Kevin; Olivares, Eric; Clark, Tyson A.; Korlach, Jonas; Turner, Stephen W.

    2010-01-01

    We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions. PMID:20453866

  1. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    PubMed Central

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

  2. Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

    PubMed

    Christensen, Laura A; Wang, Hong; Van Houten, Bennett; Vasquez, Karen M

    2008-12-01

    Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification. PMID:18996898

  3. DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides

    PubMed Central

    Laos, Roberto; Thomson, J. Michael; Benner, Steven A.

    2014-01-01

    DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic “alphabet.” This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution. PMID:25400626

  4. Homology-directed Fanconi anemia pathway crosslink repair is dependent on DNA replication

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Perrouault, Loïc; Giovannangeli, Carine; Moynahan, Mary Ellen; Barchi, Marco; Brunet, Erika; Jasin, Maria

    2012-01-01

    Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand crosslinks (ICLs) in mammalian cells. Cells from Fanconi anemia (FA) patients are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here, we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate. PMID:21423196

  5. Chase the direct impact of rainfall into groundwater in Mt. Fuji from multiple analyses including microbial DNA

    NASA Astrophysics Data System (ADS)

    Kato, Kenji; Sugiyama, Ayumi; Nagaosa, Kazuyo; Tsujimura, Maki

    2016-04-01

    A huge amount of groundwater is stored in subsurface environment of Mt. Fuji, the largest volcanic mountain in Japan. Based on the concept of piston flow transport of groundwater an apparent residence time was estimated to ca. 30 years by 36Cl/Cl ratio (Tosaki et al., 2011). However, this number represents an averaged value of the residence time of groundwater which had been mixed before it flushes out. We chased signatures of direct impact of rainfall into groundwater to elucidate the routes of groundwater, employing three different tracers; stable isotopic analysis (delta 18O), chemical analysis (concentration of silica) and microbial DNA analysis. Though chemical analysis of groundwater shows an averaged value of the examined water which was blended by various water with different sources and routes in subsurface environment, microbial DNA analysis may suggest the place where they originated, which may give information of the source and transport routes of the water examined. Throughout the in situ observation of four rainfall events showed that stable oxygen isotopic ratio of spring water and shallow groundwater obtained from 726m a.s.l. where the average recharge height of rainfall was between 1500 and 1800 m became higher than the values before a torrential rainfall, and the concentration of silica decreased after this event when rainfall exceeded 300 mm in precipitation of an event. In addition, the density of Prokaryotes in spring water apparently increased. Those changes did not appear when rainfall did not exceed 100 mm per event. Thus, findings shown above indicated a direct impact of rainfall into shallow groundwater, which appeared within a few weeks of torrential rainfall in the studied geological setting. In addition, increase in the density of Archaea observed at deep groundwater after the torrential rainfall suggested an enlargement of the strength of piston flow transport through the penetration of rainfall into deep groundwater. This finding was

  6. The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α.

    PubMed

    Han, Ting; Goralski, Maria; Capota, Emanuela; Padrick, Shae B; Kim, Jiwoong; Xie, Yang; Nijhawan, Deepak

    2016-07-01

    CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer cells, but not in normal cells, through an unknown mechanism. We used a forward-genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1(R)). Introduction of one of these mutations into cancer cells by CRISPR-Cas9 genome editing conferred CD437 resistance, demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by the identified POLA1 mutations, supporting POLA1 as the direct antitumor target of CD437. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 that is consistent with a direct binding interaction. The discovery of POLA1 as the direct anticancer target for CD437 has the potential to catalyze the development of CD437 into an anticancer therapeutic. PMID:27182663

  7. PE-Swab Direct STR Amplification of Forensic Touch DNA Samples.

    PubMed

    Liu, Jason Y

    2015-05-01

    The PE-Swab direct STR amplification workflow was developed to process low-level "touch DNA" samples. In this workflow, a forensic sample is first collected on a 4-mm PE-Swab (a novel sample collection device); two 2-mm punches containing collected samples are then generated from the PE-Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE-Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE-Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE-swab workflow is 3 times higher than that from the conventional workflow with both low-level single source and two-contributor mixture samples tested in this study. PMID:25684449

  8. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  9. DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS

    EPA Science Inventory

    Rapid, sensitive and simple assays for radiation- and chemically-induced DNA damage can be of significant benefit to a number of fields including radiation biology, clinical research, and environmental monitoring. Although temperature-induced DNA strand separation has been use...

  10. Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

    PubMed

    Takahashi, Shunsuke; Motooka, Shinya; Usui, Tomohiro; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2015-03-17

    Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA. PMID:25697222

  11. Monitoring DNA Contamination in Handled vs. Directly Excavated Ancient Human Skeletal Remains

    PubMed Central

    Pilli, Elena; Modi, Alessandra; Serpico, Ciro; Achilli, Alessandro; Lancioni, Hovirag; Lippi, Barbara; Bertoldi, Francesca; Gelichi, Sauro; Lari, Martina; Caramelli, David

    2013-01-01

    Bones, teeth and hair are often the only physical evidence of human or animal presence at an archaeological site; they are also the most widely used sources of samples for ancient DNA (aDNA) analysis. Unfortunately, the DNA extracted from ancient samples, already scarce and highly degraded, is widely susceptible to exogenous contaminations that can affect the reliability of aDNA studies. We evaluated the molecular effects of sample handling on five human skeletons freshly excavated from a cemetery dated between the 11 to the 14th century. We collected specimens from several skeletal areas (teeth, ribs, femurs and ulnas) from each individual burial. We then divided the samples into two different sets: one labeled as “virgin samples” (i.e. samples that were taken by archaeologists under contamination-controlled conditions and then immediately sent to the laboratory for genetic analyses), and the second called “lab samples”(i.e. samples that were handled without any particular precautions and subject to normal washing, handling and measuring procedures in the osteological lab). Our results show that genetic profiles from “lab samples” are incomplete or ambiguous in the different skeletal areas while a different outcome is observed in the “virgin samples” set. Generally, all specimens from different skeletal areas in the exception of teeth present incongruent results between “lab” and “virgin” samples. Therefore teeth are less prone to contamination than the other skeletal areas we analyzed and may be considered a material of choice for classical aDNA studies. In addition, we showed that bones can also be a good candidate for human aDNA analysis if they come directly from the excavation site and are accompanied by a clear taphonomic history. PMID:23372650

  12. Functionalized 2'-amino-alpha-L-LNA: directed positioning of intercalators for DNA targeting.

    PubMed

    Kumar, T Santhosh; Madsen, Andreas S; Østergaard, Michael E; Sau, Sujay P; Wengel, Jesper; Hrdlicka, Patrick J

    2009-02-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting. PMID:19108636

  13. Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting

    PubMed Central

    Kumar, T. Santhosh; Madsen, Andreas S.; Østergaard, Michael E.; Sau, Sujay P.; Wengel, Jesper; Hrdlicka, Patrick J.

    2010-01-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting. PMID:19108636

  14. [DNA and chemical analyses of commercial fly agaric-related products].

    PubMed

    Maruyama, Takuro; Kawahara, Nobuo; Fukiharu, Toshimitsu; Yokoyama, Kazumasa; Makino, Yukiko; Goda, Yukihiro

    2005-04-01

    Since June 6, 2002, psilocin and psilocybin-containing fungi (commonly called "magic mushrooms") have been regulated by the Narcotics and Psychotropics Control Law in Japan. However, various fly agaric-related products are now entering the Japanese market via the internet. In this study, fly agaric-related products available in this way were investigated for raw materials by DNA analysis and for additives by chemical analysis. Nucleotide sequence analysis of the mitochondrial 12S rDNA region suggested that these fly agaric-related products originate from A. muscaria or A. muscaria var. persicina. Furthermore, they were classified into three strains based on the ITS2-LSU nucleotide sequence. Harmine derivatives and/or tryptamine derivatives were detected in some of these products by LC/MS analysis. In accordance with this, the matK gene of Peganum harmala was found in all of the harmine derivative-containing samples. PMID:16018591

  15. Direct electrical and mechanical characterization of in situ generated DNA between the tips of silicon nanotweezers (SNT).

    PubMed

    Karsten, Stanislav L; Kumemura, Momoko; Jalabert, Laurent; Lafitte, Nicolas; Kudo, Lili C; Collard, Dominique; Fujita, Hiroyuki

    2016-05-24

    Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection. PMID:27161663

  16. Direct observation of the hole protonation state and hole localization site in DNA-oligomers

    PubMed Central

    Adhikary, Amitava; Khanduri, Deepti; Sevilla, Michael D.

    2009-01-01

    In this work, it is shown that the incorporation of an 8-deuteroguanine (G*) moiety in DNA-oligomers allows for direct determination at 77 K of (i) the location of holes (i.e., the radical site) within dsDNA at specific base sites, even within stacks of G, as well as (ii) the protonation state of the hole at that site. These findings are based on our work and demonstrate that selective deuteration at C-8 on guanine moiety in dGuo results in an ESR signal from the guanine cation radical (G*•+) which is easily distinguishable from that of the undeuterated guanine cation radical (G•+). G*•+ is also found to be easily distinguishable from its conjugate base, the N1-deprotonated radical, G*(−H)•. Our ESR results clearly establish that at 77 K (i) one-electron oxidized guanine in double stranded DNA-oligomers exists as the deprotonated neutral radical G(−H)• as a result of facile proton transfer to the hydrogen bonded cytosine, and (ii) the hole is preferentially located at the 5′-end in several ds DNA-oligomers with a GGG sequence. PMID:19469533

  17. A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

    PubMed Central

    Park, Ah Young; Jergic, Slobodan; Politis, Argyris; Ruotolo, Brandon T.; Hirshberg, Daniel; Jessop, Linda L.; Beck, Jennifer L.; Barsky, Daniel; O’Donnell, Mike; Dixon, Nicholas E.; Robinson, Carol V.

    2016-01-01

    SUMMARY Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (τ/γ) proteins, δ, and δ′, which together form an asymmetric pentameric ring that also interacts with ψχ. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that γ exists exclusively as a stable homotetramer, while τ is in a monomer-dimer-trimer-tetramer equilibrium. δ′ plays a direct role in the assembly as a τ/γ oligomer breaker, thereby facilitating incorporation of lower oligomers. With δ′, both δ and ψχ stabilize the trimeric form of DnaX, thus completing the assembly. When τ and γ are present simultaneously, mimicking the situation in vivo, subunit exchange between τ and γ tetramers occurs rapidly to form heterocomplexes but is retarded when δδ′ is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed. PMID:20223211

  18. Directing folding pathways for multi-component DNA origami nanostructures with complex topology

    NASA Astrophysics Data System (ADS)

    Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

    2016-05-01

    Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

  19. DNA Concentration-Dependent Dissociation of EcoRI: Direct Transfer or Reaction during Hopping

    PubMed Central

    Sidorova, Nina Y.; Scott, Thomas; Rau, Donald C.

    2013-01-01

    Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer. PMID:23528089

  20. A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously.

    PubMed

    An, Yingfeng; Lv, Anguo; Wu, Wenfang

    2010-06-25

    Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation. PMID:20471367

  1. Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: Implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells

    NASA Astrophysics Data System (ADS)

    Deering, R. A.

    1994-10-01

    The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation.

  2. Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells.

    PubMed

    Deering, R A

    1994-10-01

    The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation. PMID:11539974

  3. Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

    PubMed Central

    Graham, James E.; Sherratt, David J.; Szczelkun, Mark D.

    2010-01-01

    FtsK is a homohexameric, RecA-like dsDNA translocase that plays a key role in bacterial chromosome segregation. The FtsK regulatory γ-subdomain determines directionality of translocation through its interaction with specific 8 base pair chromosomal sequences [(KOPS); FtsK Orienting / Polarizing Sequence(s)] that are cooriented with the direction of replication in the chromosome. We use millisecond-resolution ensemble translocation and ATPase assays to analyze the assembly, initiation, and translocation of FtsK. We show that KOPS are used to initiate new translocation events rather than reorient existing ones. By determining kinetic parameters, we show sigmoidal dependences of translocation and ATPase rates on ATP concentration that indicate sequential cooperative coupling of ATP hydrolysis to DNA motion. We also estimate the ATP coupling efficiency of translocation to be 1.63–2.11 bp of dsDNA translocated/ATP hydrolyzed. The data were used to derive a model for the assembly, initiation, and translocation of FtsK hexamers. PMID:21048089

  4. Electron-beam lithography and molecular liftoff for directed attachment of DNA nanostructures on silicon: top-down meets bottom-up.

    PubMed

    Pillers, Michelle; Goss, Valerie; Lieberman, Marya

    2014-06-17

    CONSPECTUS: Our work on lithographic patterning of DNA nanostructures was inspired by a collaboration on molecular electronic devices known as quantum-dot cellular automata or QCA. QCA is a paradigm for computation in which information is transmitted and processed through the interaction of coupled electrical charges or magnetic dipoles. We began to explore the idea of molecular scale QCA and found that ab initio methods, a thermodynamic Ising model, and larger scale circuit design work suggested that circuits that did computationally interesting things could function at room temperature if made from molecular QCA cells of chemically reasonable design. But how could the QCA cells be patterned to form the complex arrays needed for computationally interesting circuitry, and how could those arrays of molecular circuitry be integrated with conventional electronic inputs and outputs? Top-down methods lacked the spatial resolution and high level of parallelism needed to make molecular circuits. Bottom-up chemical synthesis lacked the ability to fabricate arbitrary and heterogeneous structures tens to hundreds of nanometers in size. Chemical self-assembly at the time could produce structures in the right size scale, but was limited to homogeneous arrays. A potential solution to this conundrum was just being demonstrated in the late 1990s and early 2000s: DNA nanostructures self-assembled from oligonucleotides, whose high information density could handle the creation of arbitrary structures and chemical inhomogeneity. Our group became interested in whether DNA nanostructures could function as self-assembling circuit boards for electrical or magnetic QCA systems. This Account focuses on what we learned about the interactions of DNA nanostructures with silicon substrates and, particularly, on how electron-beam lithography could be used to direct the binding of DNA nanostructures on a variety of functional substrates. PMID:24716716

  5. Connecting the dots of RNA-directed DNA methylation in Arabidopsis thaliana.

    PubMed

    Costa-Nunes, Pedro; Vitins, Alexa; Pontes, Olga

    2014-06-01

    Noncoding RNAs are the rising stars of genome regulation and are crucial to an organism's metabolism, development, and defense. One of their most notable functions is its ability to direct epigenetic modifications through small RNA molecules to specific genomic regions, ensuring transcriptional regulation, proper genome organization, and maintenance of genome integrity. Here, we review the current knowledge of the spatial organization of the Arabidopsis thaliana RNA-directed DNA methylation pathway within the cell nucleus, which, while known to be essential for the proper establishment of epigenetic modifications, remains poorly understood. We will also discuss possible future cytological approaches that have the potential of unveiling functional insights into how small RNA-directed epigenetics is regulated through the spatiotemporal regulation of its major components within the cell. PMID:24846724

  6. Direct mechanical stimulation of tip links in hair cells through DNA tethers

    PubMed Central

    Basu, Aakash; Lagier, Samuel; Vologodskaia, Maria; Fabella, Brian A; Hudspeth, AJ

    2016-01-01

    Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels. DOI: http://dx.doi.org/10.7554/eLife.16041.001 PMID:27331611

  7. The possible roles of water in the prebiotic chemical evolution of DNA.

    PubMed

    Cui, Shuxun

    2010-09-21

    There is no doubt that water is pivotal to life. Yet, as the emergence of life is still a big challenge in science, the detailed involvement of water in that process is not well recognized. Following the clues provided by recent single-molecule studies on DNA, we attempt to elucidate the possible roles of water in the prebiotic chemical evolution. Water has long been recognized as an important reactant in the Miller-Urey experiment and then as the only solvent of the primitive soup. Besides that, water also played a vital role in the prebiotic chemical evolution: water is the important criterion in the combinatorial library screening for self-assembling macromolecules. With this notion, the uniformity of biochemistry for all terrestrial life may be explained. A possible roadmap from the inorganic world to the origin of life is also discussed. PMID:20577681

  8. RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

    PubMed Central

    Li, Qing; Gent, Jonathan I.; Zynda, Greg; Song, Jawon; Makarevitch, Irina; Hirsch, Cory D.; Hirsch, Candice N.; Dawe, R. Kelly; Madzima, Thelma F.; McGinnis, Karen M.; Lisch, Damon; Schmitz, Robert J.; Vaughn, Matthew W.; Springer, Nathan M.

    2015-01-01

    The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes. PMID:26553984

  9. The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity*

    PubMed Central

    Aria, Valentina; De Felice, Mariarita; Di Perna, Roberta; Uno, Shuji; Masai, Hisao; Syväoja, Juhani E.; van Loon, Barbara; Hübscher, Ulrich; Pisani, Francesca M.

    2013-01-01

    The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ϵ. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ϵ directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ϵ bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork. PMID:23511638

  10. The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

    PubMed Central

    Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-01-01

    Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system