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Sample records for synthetic dna sequences

  1. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    Science.gov (United States)

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  2. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

    Science.gov (United States)

    Ogata, Jun; Uchiyama, Nahoko; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2013-04-10

    In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  4. Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications

    Energy Technology Data Exchange (ETDEWEB)

    Torella, JP; Lienert, F; Boehm, CR; Chen, JH; Way, JC; Silver, PA

    2014-08-07

    Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies-for example, repeated terminator and insulator sequences-that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)-guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2-3 d. If the DNA parts must be generated from scratch, an additional 2-5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.

  5. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA

    DEFF Research Database (Denmark)

    Minero, Gabriel Khose Antonio; Nogueira, Catarina; Rizzi, Giovanni

    2017-01-01

    magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic...... nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP...... amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we...

  6. Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

    Science.gov (United States)

    Minero, Gabriel Antonio S; Nogueira, Catarina; Rizzi, Giovanni; Tian, Bo; Fock, Jeppe; Donolato, Marco; Strömberg, Mattias; Hansen, Mikkel F

    2017-09-08

    We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

  7. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  8. DNA recognition by synthetic constructs.

    Science.gov (United States)

    Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

    2011-09-05

    The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Gomphid DNA sequence data

    Data.gov (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  10. DNA Sequencing apparatus

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  11. Metric representation of DNA sequences.

    Science.gov (United States)

    Wu, Z B

    2000-07-01

    A metric representation of DNA sequences is borrowed from symbolic dynamics. In view of this method, the pattern seen in the chaos game representation of DNA sequences is explained as the suppression of certain nucleotide strings in the DNA sequences. Frequencies of short nucleotide strings and suppression of the shortest ones in the DNA sequences can be determined by using the metric representation.

  12. Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'-CGG-3')17 oligodeoxyribonucleotide

    DEFF Research Database (Denmark)

    Behn-Krappa, A; Mollenhauer, J; Doerfler, W

    1993-01-01

    The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy (Kenn...

  13. Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'-CGG-3')17 oligodeoxyribonucleotide

    DEFF Research Database (Denmark)

    Behn-Krappa, A; Mollenhauer, J; Doerfler, W

    1993-01-01

    The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy...

  14. Evolution of DNA sequencing.

    Science.gov (United States)

    Tipu, Hamid Nawaz; Shabbir, Ambreen

    2015-03-01

    Sanger and coworkers introduced DNA sequencing in 1970s for the first time. It principally relied on termination of growing nucleotide chain when a dideoxythymidine triphosphate (ddTTP) was inserted in it. Detection of terminated sequences was done radiographically on Polyacrylamide Gel Electrophoresis (PAGE). Improvements that have evolved over time in original Sanger sequencing include replacement of radiography with fluorescence, use of separate fluorescent markers for each nucleotide, use of capillary electrophoresis instead of polyacrylamide gel electrophoresis and then introduction of capillary array electrophoresis. However, this technique suffered from few inherent limitations like decreased sensitivity for low level mutant alleles, complexities in analyzing highly polymorphic regions like Major Histocompatibility Complex (MHC) and high DNA concentrations required. Several Next Generation Sequencing (NGS) technologies have been introduced by Roche, Illumina and other commercial manufacturers that tend to overcome Sanger sequencing limitations and have been reviewed. Introduction of NGS in clinical research and medical diagnostics is expected to change entire diagnostic approach. These include study of cancer variants, detection of minimal residual disease, exome sequencing, detection of Single Nucleotide Polymorphisms (SNPs) and their disease association, epigenetic regulation of gene expression and sequencing of microorganisms genome.

  15. The Dynamics of DNA Sequencing.

    Science.gov (United States)

    Morvillo, Nancy

    1997-01-01

    Describes a paper-and-pencil activity that helps students understand DNA sequencing and expands student understanding of DNA structure, replication, and gel electrophoresis. Appropriate for advanced biology students who are familiar with the Sanger method. (DDR)

  16. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  17. Graphene nanodevices for DNA sequencing

    Science.gov (United States)

    Heerema, Stephanie J.; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.

  18. Adenoviral DNA replication: DNA sequences and enzymes required for initiation in vitro

    International Nuclear Information System (INIS)

    Stillman, B.W.; Tamanoi, F.

    1983-01-01

    In this paper evidence is provided that the 140,000-dalton DNA polymerase is encoded by the adenoviral genome and is required for the initiation of DNA replication in vitro. The DNA sequences in the template DNA that are required for the initiation of replication have also been identified, using both plasmid DNAs and synthetic oligodeoxyribonucleotides. 48 references, 7 figures, 1 table

  19. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  20. Duplication in DNA Sequences

    Science.gov (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  1. Graphene nanodevices for DNA sequencing

    NARCIS (Netherlands)

    Heerema, S.J.; Dekker, C.

    2016-01-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with

  2. Image analysis for DNA sequencing

    International Nuclear Information System (INIS)

    Palaniappan, K.; Huang, T.S.

    1991-01-01

    This paper reports that there is a great deal of interest in automating the process of DNA (deoxyribonucleic acid) sequencing to support the analysis of genomic DNA such as the Human and Mouse Genome projects. In one class of gel-based sequencing protocols autoradiograph images are generated in the final step and usually require manual interpretation to reconstruct the DNA sequence represented by the image. The need to handle a large volume of sequence information necessitates automation of the manual autoradiograph reading step through image analysis in order to reduce the length of time required to obtain sequence data and reduce transcription errors. Various adaptive image enhancement, segmentation and alignment methods were applied to autoradiograph images. The methods are adaptive to the local characteristics of the image such as noise, background signal, or presence of edges. Once the two-dimensional data is converted to a set of aligned one-dimensional profiles waveform analysis is used to determine the location of each band which represents one nucleotide in the sequence. Different classification strategies including a rule-based approach are investigated to map the profile signals, augmented with the original two-dimensional image data as necessary, to textual DNA sequence information

  3. DNA Sequencing Sensors: An Overview

    Directory of Open Access Journals (Sweden)

    Jose Antonio Garrido-Cardenas

    2017-03-01

    Full Text Available The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

  4. Sequence analysis of Leukemia DNA

    Science.gov (United States)

    Nacong, Nasria; Lusiyanti, Desy; Irawan, Muhammad. Isa

    2018-03-01

    Cancer is a very deadly disease, one of which is leukemia disease or better known as blood cancer. The cancer cell can be detected by taking DNA in laboratory test. This study focused on local alignment of leukemia and non leukemia data resulting from NCBI in the form of DNA sequences by using Smith-Waterman algorithm. SmithWaterman algorithm was invented by TF Smith and MS Waterman in 1981. These algorithms try to find as much as possible similarity of a pair of sequences, by giving a negative value to the unequal base pair (mismatch), and positive values on the same base pair (match). So that will obtain the maximum positive value as the end of the alignment, and the minimum value as the initial alignment. This study will use sequences of leukemia and 3 sequences of non leukemia.

  5. DNA Sequencing by Capillary Electrophoresis

    Science.gov (United States)

    Karger, Barry L.; Guttman, Andras

    2009-01-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

  6. A Demonstration of Automated DNA Sequencing.

    Science.gov (United States)

    Latourelle, Sandra; Seidel-Rogol, Bonnie

    1998-01-01

    Details a simulation that employs a paper-and-pencil model to demonstrate the principles behind automated DNA sequencing. Discusses the advantages of automated sequencing as well as the chemistry of automated DNA sequencing. (DDR)

  7. Apparatus for improved DNA sequencing

    Science.gov (United States)

    Douthart, Richard J.; Crowell, Shannon L.

    1996-01-01

    This invention is a means for the rapid sequencing of DNA samples. More specifically, it consists of a new design direct blotting electrophoresis unit. The DNA sequence is deposited on a membrane attached to a rotating drum. Initial data compaction is facilitated by the use of a machined multi-channeled plate called a ribbon channel plate. Each channel is an isolated mini gel system much like a gel filled capillary. The system as a whole, however, is in a slab gel like format with the advantages of uniformity and easy reusability. The system can be used in different embodiments. The drum system is unique in that after deposition the drum rotates the deposited DNA into a large non-buffer open space where processing and detection can occur. The drum can also be removed in toto to special workstations for downstream processing, multiplexing and detection.

  8. The sequence of sequencers: The history of sequencing DNA

    Science.gov (United States)

    Heather, James M.; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. PMID:26554401

  9. Multiplexed Sequence Encoding: A Framework for DNA Communication

    Science.gov (United States)

    Zakeri, Bijan; Carr, Peter A.; Lu, Timothy K.

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication—data encoding, data transfer & data extraction—and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system—Multiplexed Sequence Encoding (MuSE)—that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  10. Channel plate for DNA sequencing

    Science.gov (United States)

    Douthart, Richard J.; Crowell, Shannon L.

    1998-01-01

    This invention is a channel plate that facilitates data compaction in DNA sequencing. The channel plate has a length, a width and a thickness, and further has a plurality of channels that are parallel. Each channel has a depth partially through the thickness of the channel plate. Additionally an interface edge permits electrical communication across an interface through a buffer to a deposition membrane surface.

  11. Entropic fluctuations in DNA sequences

    Science.gov (United States)

    Thanos, Dimitrios; Li, Wentian; Provata, Astero

    2018-03-01

    The Local Shannon Entropy (LSE) in blocks is used as a complexity measure to study the information fluctuations along DNA sequences. The LSE of a DNA block maps the local base arrangement information to a single numerical value. It is shown that despite this reduction of information, LSE allows to extract meaningful information related to the detection of repetitive sequences in whole chromosomes and is useful in finding evolutionary differences between organisms. More specifically, large regions of tandem repeats, such as centromeres, can be detected based on their low LSE fluctuations along the chromosome. Furthermore, an empirical investigation of the appropriate block sizes is provided and the relationship of LSE properties with the structure of the underlying repetitive units is revealed by using both computational and mathematical methods. Sequence similarity between the genomic DNA of closely related species also leads to similar LSE values at the orthologous regions. As an application, the LSE covariance function is used to measure the evolutionary distance between several primate genomes.

  12. Perspectives in Biochemistry: Methods for DNA Sequencing.

    Science.gov (United States)

    Wood, Anne T.

    1984-01-01

    Describes two frequently used DNA sequencing methods: Sander's enzymatic dideoxy method and Maxam and Gilbert's chemical sequencing method. Indicates that studying these methods provides students with knowledge of the chemical structure of DNA and how DNA sequence data are obtained. (JN)

  13. DNA sequencing technologies: 2006-2016.

    Science.gov (United States)

    Mardis, Elaine R

    2017-02-01

    Recent advances in the field of genomics have largely been due to the ability to sequence DNA at increasing throughput and decreasing cost. DNA sequencing was first introduced in 1977, and next-generation sequencing technologies have been available only during the past decade, but the diverse experiments and corresponding analyses facilitated by these techniques have transformed biological and biomedical research. Here, I review developments in DNA sequencing technologies over the past 10 years and look to the future for further applications.

  14. Programmable in vivo selection of arbitrary DNA sequences.

    Directory of Open Access Journals (Sweden)

    Tuval Ben Yehezkel

    Full Text Available The extraordinary fidelity, sensory and regulatory capacity of natural intracellular machinery is generally confined to their endogenous environment. Nevertheless, synthetic bio-molecular components have been engineered to interface with the cellular transcription, splicing and translation machinery in vivo by embedding functional features such as promoters, introns and ribosome binding sites, respectively, into their design. Tapping and directing the power of intracellular molecular processing towards synthetic bio-molecular inputs is potentially a powerful approach, albeit limited by our ability to streamline the interface of synthetic components with the intracellular machinery in vivo. Here we show how a library of synthetic DNA devices, each bearing an input DNA sequence and a logical selection module, can be designed to direct its own probing and processing by interfacing with the bacterial DNA mismatch repair (MMR system in vivo and selecting for the most abundant variant, regardless of its function. The device provides proof of concept for programmable, function-independent DNA selection in vivo and provides a unique example of a logical-functional interface of an engineered synthetic component with a complex endogenous cellular system. Further research into the design, construction and operation of synthetic devices in vivo may lead to other functional devices that interface with other complex cellular processes for both research and applied purposes.

  15. Method for sequencing DNA base pairs

    Science.gov (United States)

    Sessler, Andrew M.; Dawson, John

    1993-01-01

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source.

  16. DNA Sequencing in Undergraduate Laboratory Courses.

    Science.gov (United States)

    Hamilton, Robert G.

    1997-01-01

    Discusses strategies to duplicate current research protocols using biochemical methods of analysis. Describes the use of the Silver Sequence kit that provides a technically simple and relatively inexpensive DNA sequencing exercise. (JRH)

  17. "First generation" automated DNA sequencing technology.

    Science.gov (United States)

    Slatko, Barton E; Kieleczawa, Jan; Ju, Jingyue; Gardner, Andrew F; Hendrickson, Cynthia L; Ausubel, Frederick M

    2011-10-01

    Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines. © 2011 by John Wiley & Sons, Inc.

  18. DNA meets synthetic polymers—highly versatile hybrid materials

    NARCIS (Netherlands)

    Alemdaroglu, Fikri E.; Herrmann, Andreas

    2007-01-01

    The combination of synthetic polymers and DNA has provided biologists, chemists and materials scientists with a fascinating new hybrid material. The challenges in preparing these molecular chimeras were overcome by different synthetic strategies that rely on coupling the nucleic acid moiety and the

  19. Designing Uniquely Addressable Bio-orthogonal Synthetic Scaffolds for DNA and RNA Origami.

    Science.gov (United States)

    Kozyra, Jerzy; Ceccarelli, Alessandro; Torelli, Emanuela; Lopiccolo, Annunziata; Gu, Jing-Ying; Fellermann, Harold; Stimming, Ulrich; Krasnogor, Natalio

    2017-07-21

    Nanotechnology and synthetic biology are rapidly converging, with DNA origami being one of the leading bridging technologies. DNA origami was shown to work well in a wide array of biotic environments. However, the large majority of extant DNA origami scaffolds utilize bacteriophages or plasmid sequences thus severely limiting its future applicability as a bio-orthogonal nanotechnology platform. In this paper we present the design of biologically inert (i.e., "bio-orthogonal") origami scaffolds. The synthetic scaffolds have the additional advantage of being uniquely addressable (unlike biologically derived ones) and hence are better optimized for high-yield folding. We demonstrate our fully synthetic scaffold design with both DNA and RNA origamis and describe a protocol to produce these bio-orthogonal and uniquely addressable origami scaffolds.

  20. Sequencing of adenine in DNA by scanning tunneling microscopy

    Science.gov (United States)

    Tanaka, Hiroyuki; Taniguchi, Masateru

    2017-08-01

    The development of DNA sequencing technology utilizing the detection of a tunnel current is important for next-generation sequencer technologies based on single-molecule analysis technology. Using a scanning tunneling microscope, we previously reported that dI/dV measurements and dI/dV mapping revealed that the guanine base (purine base) of DNA adsorbed onto the Cu(111) surface has a characteristic peak at V s = -1.6 V. If, in addition to guanine, the other purine base of DNA, namely, adenine, can be distinguished, then by reading all the purine bases of each single strand of a DNA double helix, the entire base sequence of the original double helix can be determined due to the complementarity of the DNA base pair. Therefore, the ability to read adenine is important from the viewpoint of sequencing. Here, we report on the identification of adenine by STM topographic and spectroscopic measurements using a synthetic DNA oligomer and viral DNA.

  1. Using DNA looping to measure sequence dependent DNA elasticity

    Science.gov (United States)

    Kandinov, Alan; Raghunathan, Krishnan; Meiners, Jens-Christian

    2012-10-01

    We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

  2. Multiple tag labeling method for DNA sequencing

    Science.gov (United States)

    Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

    1995-01-01

    A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

  3. Nuclear DNA sequences from late Pleistocene megafauna.

    Science.gov (United States)

    Greenwood, A D; Capelli, C; Possnert, G; Pääbo, S

    1999-11-01

    We report the retrieval and characterization of multi- and single-copy nuclear DNA sequences from Alaskan and Siberian mammoths (Mammuthus primigenius). In addition, a nuclear copy of a mitochondrial gene was recovered. Furthermore, a 13,000-year-old ground sloth and a 33,000-year-old cave bear yielded multicopy nuclear DNA sequences. Thus, multicopy and single-copy genes can be analyzed from Pleistocene faunal remains. The results also show that under some circumstances, nucleotide sequence differences between alleles found within one individual can be distinguished from DNA sequence variation caused by postmortem DNA damage. The nuclear sequences retrieved from the mammoths suggest that mammoths were more similar to Asian elephants than to African elephants.

  4. Synthetic Nucleotides as Probes of DNA Polymerase Specificity

    Directory of Open Access Journals (Sweden)

    Jason M. Walsh

    2012-01-01

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

  5. EGNAS: an exhaustive DNA sequence design algorithm

    Directory of Open Access Journals (Sweden)

    Kick Alfred

    2012-06-01

    Full Text Available Abstract Background The molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences. Results The algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented. Conclusions We developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use at http://www.chm.tu-dresden.de/pc6/EGNAS.

  6. An approach to sequence DNA without tagging

    Science.gov (United States)

    Niu, Sanjun; Saraf, Ravi F.

    2002-10-01

    Microarray technology is playing an increasingly important role in biology and medicine and its application to genomics for gene expression analysis has already reached the market with a variety of commercially available instruments. In these combinatorial analysis methods, known probe single-strand DNA (ssDNA) 'primers' are attached in clusters of typically 100 µm × 100 µm pixels. Each pixel of the array has a slightly different sequence. On exposure to 'unknown' target ssDNA, the pixels with the right complementary probe ssDNA sequence convert to double-stranded DNA (dsDNA) by a hybridization reaction. To transduct the conversion of the pixel to dsDNA, the target ssDNA is labelled with a photoluminescent tag during the polymerase chain reaction (PCR) amplification process. Due to the statistical distribution of the tags in the target ssDNA, it becomes significantly difficult to implement these methods as a diagnostic tool in a pathology laboratory. A method to sequence DNA without tagging the molecule is developed. The fabrication process is compatible with current microelectronics and (emerging) soft-material fabrication technologies, allowing the method to be integrable with micro-electromechanical systems (MEMS) and lab-on-a-chip devices. An estimated sensitivity of 10-12 g on a 1 cm2 device area is obtained.

  7. Chromatid interchanges at intrachromosomal telomeric DNA sequences

    International Nuclear Information System (INIS)

    Fernandez, J.L.; Vazquez-Gundin, F.; Bilbao, A.; Gosalvez, J.; Goyanes, V.

    1997-01-01

    Chinese hamster Don cells were exposed to X-rays, mitomycin C and teniposide (VM-26) to induce chromatid exchanges (quadriradials and triradials). After fluorescence in situ hybridization (FISH) of telomere sequences it was found that interstitial telomere-like DNA sequence arrays presented around five times more breakage-rearrangements than the genome overall. This high recombinogenic capacity was independent of the clastogen, suggesting that this susceptibility is not related to the initial mechanisms of DNA damage. (author)

  8. Mitochondrial DNA sequence variation in Hippopotamus amphibius ...

    African Journals Online (AJOL)

    Mitochondrial DNA sequence variation in Hippopotamus amphibius from Kruger National Park, Republic of South Africa. ... A test of the hypothesis that calves are more likely to share a mtDNA haplotype with an adult female in the same herd than an adult female from a different herd was not significant. Keywords: ...

  9. Mitochondrial DNA sequence evolution in shorebird populations

    NARCIS (Netherlands)

    Wenink, P.W.

    1994-01-01

    This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons

  10. On site DNA barcoding by nanopore sequencing.

    Directory of Open Access Journals (Sweden)

    Michele Menegon

    Full Text Available Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet's biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities.

  11. DNA sequencing using fluorescence background electroblotting membrane

    Science.gov (United States)

    Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

    1992-05-12

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

  12. DNA sequencing using fluorescence background electroblotting membrane

    Science.gov (United States)

    Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

    1992-01-01

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

  13. Nanogrid rolling circle DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Church, George M.; Porreca, Gregory J.; Shendure, Jay; Rosenbaum, Abraham Meir

    2017-04-18

    The present invention relates to methods for sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size, including synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region, immobilizing the concatemer to the specific region to make an immobilized concatemer, and sequencing the immobilized concatemer.

  14. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  15. Nanopore-CMOS Interfaces for DNA Sequencing.

    Science.gov (United States)

    Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

    2016-08-06

    DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces.

  16. RANDNA: a random DNA sequence generator.

    Science.gov (United States)

    Piva, Francesco; Principato, Giovanni

    2006-01-01

    Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

  17. Compressing DNA sequence databases with coil

    Directory of Open Access Journals (Sweden)

    Hendy Michael D

    2008-05-01

    Full Text Available Abstract Background Publicly available DNA sequence databases such as GenBank are large, and are growing at an exponential rate. The sheer volume of data being dealt with presents serious storage and data communications problems. Currently, sequence data is usually kept in large "flat files," which are then compressed using standard Lempel-Ziv (gzip compression – an approach which rarely achieves good compression ratios. While much research has been done on compressing individual DNA sequences, surprisingly little has focused on the compression of entire databases of such sequences. In this study we introduce the sequence database compression software coil. Results We have designed and implemented a portable software package, coil, for compressing and decompressing DNA sequence databases based on the idea of edit-tree coding. coil is geared towards achieving high compression ratios at the expense of execution time and memory usage during compression – the compression time represents a "one-off investment" whose cost is quickly amortised if the resulting compressed file is transmitted many times. Decompression requires little memory and is extremely fast. We demonstrate a 5% improvement in compression ratio over state-of-the-art general-purpose compression tools for a large GenBank database file containing Expressed Sequence Tag (EST data. Finally, coil can efficiently encode incremental additions to a sequence database. Conclusion coil presents a compelling alternative to conventional compression of flat files for the storage and distribution of DNA sequence databases having a narrow distribution of sequence lengths, such as EST data. Increasing compression levels for databases having a wide distribution of sequence lengths is a direction for future work.

  18. Quantum-Sequencing: Fast electronic single DNA molecule sequencing

    Science.gov (United States)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

  19. DNA Sequencing in Cultural Heritage.

    Science.gov (United States)

    Vai, Stefania; Lari, Martina; Caramelli, David

    2016-02-01

    During the last three decades, DNA analysis on degraded samples revealed itself as an important research tool in anthropology, archaeozoology, molecular evolution, and population genetics. Application on topics such as determination of species origin of prehistoric and historic objects, individual identification of famous personalities, characterization of particular samples important for historical, archeological, or evolutionary reconstructions, confers to the paleogenetics an important role also for the enhancement of cultural heritage. A really fast improvement in methodologies in recent years led to a revolution that permitted recovering even complete genomes from highly degraded samples with the possibility to go back in time 400,000 years for samples from temperate regions and 700,000 years for permafrozen remains and to analyze even more recent material that has been subjected to hard biochemical treatments. Here we propose a review on the different methodological approaches used so far for the molecular analysis of degraded samples and their application on some case studies.

  20. Design of sequence-specific DNA binding ligands that use a two-stranded peptide motif for DNA sequence recognition.

    Science.gov (United States)

    Nikolaev, V A; Grokhovsky, S L; Surovaya, A N; Leinsoo, T A; Sidorova NYu; Zasedatelev, A S; Zhuze, A L; Strahan, G A; Shafer, R H; Gursky, G V

    1996-08-01

    The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.

  1. Scintillating optical fiber detectors for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Bendali, M.; Mastrippolito, R.; Charon, Y.; Leblanc, M.; Tricoire, H.; Valentin, L. (Inst. de Physique Nucleaire, 91 - Orsay (France) Lab. de Physique Nucleaire, Univ. Paris 7, 75 (France)); Martin, B. (Lab. de Neurobiologie Cellulaire et Moleculaire, 91 - Gif-sur-Yvette (France))

    1991-12-01

    We have developed a two-dimensional detector (SOFI) for {sup 32}P emitting molecules used in molecular biology by combining scinitillating optical fibers (SOFs) and a multianode photomultiplier (MAPM). A good efficiency (15%) was obtained by suppressing the internal cross talk of the MAPM with a new electronic device. Using this improvement we are developing two new detectors using SOFs for DNA sequencing. We shall present the basic principle of these detectors and the results in efficiency and position accuracy obtained with the first prototypes. The advantage of these detectors over currently available DNA sequencers will be discussed. (orig.).

  2. A Bioluminometric Method of DNA Sequencing

    Science.gov (United States)

    Ronaghi, Mostafa; Pourmand, Nader; Stolc, Viktor; Arnold, Jim (Technical Monitor)

    2001-01-01

    Pyrosequencing is a bioluminometric single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis. In this sequencing-by-synthesis method, a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. Pyrosequencing has the advantages of accuracy, flexibility and parallel processing. It can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. In this chapter, the use of this technique for different applications is discussed.

  3. De Novo metabolic engineering and the promise of synthetic DNA.

    Science.gov (United States)

    Klein-Marcuschamer, Daniel; Yadav, Vikramaditya G; Ghaderi, Adel; Stephanopoulos, Gregory N

    2010-01-01

    -expressing single or multiple genes using recombinant DNA, and intervention targets were predominantly selected based on knowledge of the stoichiometry, kinetics, and regulation of the pathway of interest. However, the distributive nature of metabolic control, as opposed to the existence of a single rate-limiting step, predicates the controlled expression of multiple enzymes in several coordinated pathways to achieve the desired flux, and, as such, simple strategies involving either deleting or over-expressing genes are greatly limited in this context. On the other hand, the use of synthetic or modified promoters, riboswitches, tunable intergenic regions, and translation modulators such as internal ribosome entry sequences, upstream open reading frames, optimized mRNA secondary structures, and RNA silencing have been shown to be enormously conducive to achieving the fine-tuning of gene expression. These modifications to the genetic machinery of the host organism can be best achieved via the use of synthetic DNA technology, and the constant improvement in the affordability and quality of oligonucleotide synthesis suggests that these might well become the mainstay of the metabolic engineering toolbox in the years to come. The possibilities that arise with the use of synthetic oligonucleotides will be delineated herein.

  4. Nanopore Technology: A Simple, Inexpensive, Futuristic Technology for DNA Sequencing.

    Science.gov (United States)

    Gupta, P D

    2016-10-01

    In health care, importance of DNA sequencing has been fully established. Sanger's Capillary Electrophoresis DNA sequencing methodology is time consuming, cumbersome, hence become more expensive. Lately, because of its versatility DNA sequencing became house hold name, and therefore, there is an urgent need of simple, fast, inexpensive, DNA sequencing technology. In the beginning of this century efforts were made, and Nanopore DNA sequencing technology was developed; still it is infancy, nevertheless, it is the futuristic technology.

  5. Enhanced throughput for infrared automated DNA sequencing

    Science.gov (United States)

    Middendorf, Lyle R.; Gartside, Bill O.; Humphrey, Pat G.; Roemer, Stephen C.; Sorensen, David R.; Steffens, David L.; Sutter, Scott L.

    1995-04-01

    Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany; Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.

  6. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence.

    Science.gov (United States)

    Gautam, Shweta D; Chen, Jon K; Murray, Vincent

    2017-08-01

    Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

  7. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  8. Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Noremylia Mohd Bakhori

    2013-12-01

    Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  9. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    317. 2.4 cDNA sequencing and analysis. The nucleotide sequence of the cloned H. fossilis GH. cDNA was determined by Sanger's dideoxy chain termi- nation method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All other computational analysis of the GH cDNA was done using.

  10. Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

    Science.gov (United States)

    Patron, Nicola J; Orzaez, Diego; Marillonnet, Sylvestre; Warzecha, Heribert; Matthewman, Colette; Youles, Mark; Raitskin, Oleg; Leveau, Aymeric; Farré, Gemma; Rogers, Christian; Smith, Alison; Hibberd, Julian; Webb, Alex A R; Locke, James; Schornack, Sebastian; Ajioka, Jim; Baulcombe, David C; Zipfel, Cyril; Kamoun, Sophien; Jones, Jonathan D G; Kuhn, Hannah; Robatzek, Silke; Van Esse, H Peter; Sanders, Dale; Oldroyd, Giles; Martin, Cathie; Field, Rob; O'Connor, Sarah; Fox, Samantha; Wulff, Brande; Miller, Ben; Breakspear, Andy; Radhakrishnan, Guru; Delaux, Pierre-Marc; Loqué, Dominique; Granell, Antonio; Tissier, Alain; Shih, Patrick; Brutnell, Thomas P; Quick, W Paul; Rischer, Heiko; Fraser, Paul D; Aharoni, Asaph; Raines, Christine; South, Paul F; Ané, Jean-Michel; Hamberger, Björn R; Langdale, Jane; Stougaard, Jens; Bouwmeester, Harro; Udvardi, Michael; Murray, James A H; Ntoukakis, Vardis; Schäfer, Patrick; Denby, Katherine; Edwards, Keith J; Osbourn, Anne; Haseloff, Jim

    2015-10-01

    Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  11. Local Renyi entropic profiles of DNA sequences.

    Science.gov (United States)

    Vinga, Susana; Almeida, Jonas S

    2007-10-16

    In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at http://kdbio.inesc-id.pt/~svinga/ep/. The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.

  12. Local Renyi entropic profiles of DNA sequences

    Directory of Open Access Journals (Sweden)

    Vinga Susana

    2007-10-01

    Full Text Available Abstract Background In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM. Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. Results The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at http://kdbio.inesc-id.pt/~svinga/ep/. Conclusion The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.

  13. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Directory of Open Access Journals (Sweden)

    Matthias Christen

    Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  14. Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery

    Directory of Open Access Journals (Sweden)

    Kirkness Ewen

    2006-10-01

    Full Text Available Abstract Background Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromosome sequence and the search for polymorphisms therein. The genome has been only partially sequenced for one male dog, disallowing mapping of the sequence into specific chromosomes. However, by comparing the male genome sequence to the complete female dog genome sequence, candidate Y-chromosome sequence may be identified by exclusion. Results The male dog genome sequence was analysed by Blast search against the human genome to identify sequences with a best match to the human Y chromosome and to the female dog genome to identify those absent in the female genome. Candidate sequences were then tested for male specificity by PCR of five male and five female dogs. 32 sequences from the male genome, with a total length of 24 kbp, were identified as male specific, based on a match to the human Y chromosome, absence in the female dog genome and male specific PCR results. 14437 bp were then sequenced for 10 male dogs originating from Europe, Southwest Asia, Siberia, East Asia, Africa and America. Nine haplotypes were found, which were defined by 14 substitutions. The genetic distance between the haplotypes indicates that they originate from at least five wolf haplotypes. There was no obvious trend in the geographic distribution of the haplotypes. Conclusion We have identified 24159 bp of dog Y-chromosome sequence to be used for population genetic studies. We sequenced 14437 bp in a worldwide collection of dogs, identifying 14 SNPs for future SNP analyses, and

  15. DNA sequencing versus standard prenatal aneuploidy screening.

    Science.gov (United States)

    Bianchi, Diana W; Parker, R Lamar; Wentworth, Jeffrey; Madankumar, Rajeevi; Saffer, Craig; Das, Anita F; Craig, Joseph A; Chudova, Darya I; Devers, Patricia L; Jones, Keith W; Oliver, Kelly; Rava, Richard P; Sehnert, Amy J

    2014-02-27

    In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, Paneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

  16. Simulating efficiently the evolution of DNA sequences.

    Science.gov (United States)

    Schöniger, M; von Haeseler, A

    1995-02-01

    Two menu-driven FORTRAN programs are described that simulate the evolution of DNA sequences in accordance with a user-specified model. This general stochastic model allows for an arbitrary stationary nucleotide composition and any transition-transversion bias during the process of base substitution. In addition, the user may define any hypothetical model tree according to which a family of sequences evolves. The programs suggest the computationally most inexpensive approach to generate nucleotide substitutions. Either reproducible or non-repeatable simulations, depending on the method of initializing the pseudo-random number generator, can be performed. The corresponding options are offered by the interface menu.

  17. Genomic signal processing for DNA sequence clustering

    Directory of Open Access Journals (Sweden)

    Gerardo Mendizabal-Ruiz

    2018-01-01

    Full Text Available Genomic signal processing (GSP methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

  18. Genomic signal processing for DNA sequence clustering.

    Science.gov (United States)

    Mendizabal-Ruiz, Gerardo; Román-Godínez, Israel; Torres-Ramos, Sulema; Salido-Ruiz, Ricardo A; Vélez-Pérez, Hugo; Morales, J Alejandro

    2018-01-01

    Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

  19. Google matrix analysis of DNA sequences.

    Science.gov (United States)

    Kandiah, Vivek; Shepelyansky, Dima L

    2013-01-01

    For DNA sequences of various species we construct the Google matrix [Formula: see text] of Markov transitions between nearby words composed of several letters. The statistical distribution of matrix elements of this matrix is shown to be described by a power law with the exponent being close to those of outgoing links in such scale-free networks as the World Wide Web (WWW). At the same time the sum of ingoing matrix elements is characterized by the exponent being significantly larger than those typical for WWW networks. This results in a slow algebraic decay of the PageRank probability determined by the distribution of ingoing elements. The spectrum of [Formula: see text] is characterized by a large gap leading to a rapid relaxation process on the DNA sequence networks. We introduce the PageRank proximity correlator between different species which determines their statistical similarity from the view point of Markov chains. The properties of other eigenstates of the Google matrix are also discussed. Our results establish scale-free features of DNA sequence networks showing their similarities and distinctions with the WWW and linguistic networks.

  20. Google matrix analysis of DNA sequences.

    Directory of Open Access Journals (Sweden)

    Vivek Kandiah

    Full Text Available For DNA sequences of various species we construct the Google matrix [Formula: see text] of Markov transitions between nearby words composed of several letters. The statistical distribution of matrix elements of this matrix is shown to be described by a power law with the exponent being close to those of outgoing links in such scale-free networks as the World Wide Web (WWW. At the same time the sum of ingoing matrix elements is characterized by the exponent being significantly larger than those typical for WWW networks. This results in a slow algebraic decay of the PageRank probability determined by the distribution of ingoing elements. The spectrum of [Formula: see text] is characterized by a large gap leading to a rapid relaxation process on the DNA sequence networks. We introduce the PageRank proximity correlator between different species which determines their statistical similarity from the view point of Markov chains. The properties of other eigenstates of the Google matrix are also discussed. Our results establish scale-free features of DNA sequence networks showing their similarities and distinctions with the WWW and linguistic networks.

  1. What Advances Are Being Made in DNA Sequencing?

    Science.gov (United States)

    ... diagnosis in the future. For more information about DNA sequencing technologies and their use: Genetics Home Reference discusses ... illustration of the decline in the cost of DNA sequencing , including that caused by the introduction of new ...

  2. The DNA sequence of equine herpesvirus-1.

    Science.gov (United States)

    Telford, E A; Watson, M S; McBride, K; Davison, A J

    1992-07-01

    The complete DNA sequence was determined of a pathogenic British isolate of equine herpesvirus-1, a respiratory virus which can cause abortion and neurological disease. The genome is 150,223 bp in size, has a base composition of 56.7% G + C, and contains 80 open reading frames likely to encode protein. Since four open reading frames are duplicated in the major inverted repeat, two are probably expressed as a spliced mRNA, and one may contain an internal transcriptional promoter, the genome is considered to contain 76 distinct genes. The genes are arranged collinearly with those in the genomes of the two previously sequenced alphaherpesviruses, varicella-zoster virus, and herpes simplex virus type-1, and comparisons of predicted amino acid sequences allowed the functions of many equine herpesvirus 1 proteins to be assigned.

  3. Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery

    OpenAIRE

    Natanaelsson, Christian; Oskarsson, Mattias CR; Angleby, Helen; Lundeberg, Joakim; Kirkness, Ewen; Savolainen, Peter

    2006-01-01

    Abstract Background Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromo...

  4. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  5. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy

    Science.gov (United States)

    Casini, Arturo; MacDonald, James T.; Jonghe, Joachim De; Christodoulou, Georgia; Freemont, Paul S.; Baldwin, Geoff S.; Ellis, Tom

    2014-01-01

    Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. PMID:24153110

  6. Predicting DNA hybridization kinetics from sequence

    Science.gov (United States)

    Zhang, Jinny X.; Fang, John Z.; Duan, Wei; Wu, Lucia R.; Zhang, Angela W.; Dalchau, Neil; Yordanov, Boyan; Petersen, Rasmus; Phillips, Andrew; Zhang, David Yu

    2018-01-01

    Hybridization is a key molecular process in biology and biotechnology, but so far there is no predictive model for accurately determining hybridization rate constants based on sequence information. Here, we report a weighted neighbour voting (WNV) prediction algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants. To construct this algorithm we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (36 nt sub-sequences of the CYCS and VEGF genes) at temperatures ranging from 28 to 55 °C. Automated feature selection and weighting optimization resulted in a final six-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 3 with ∼91% accuracy, based on leave-one-out cross-validation. Accurate prediction of hybridization kinetics allows the design of efficient probe sequences for genomics research.

  7. Method for priming and DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Mugasimangalam, R.C.; Ulanovsky, L.E.

    1997-12-01

    A method is presented for improving the priming specificity of an oligonucleotide primer that is non-unique in a nucleic acid template which includes selecting a continuous stretch of several nucleotides in the template DNA where one of the four bases does not occur in the stretch. This also includes bringing the template DNA in contract with a non-unique primer partially or fully complimentary to the sequence immediately upstream of the selected sequence stretch. This results in polymerase-mediated differential extension of the primer in the presence of a subset of deoxyribonucleotide triphosphates that does not contain the base complementary to the base absent in the selected sequence stretch. These reactions occur at a temperature sufficiently low for allowing the extension of the non-unique primer. The method causes polymerase-mediated extension reactions in the presence of all four natural deoxyribonucleotide triphosphates or modifications. At this high temperature discrimination occurs against priming sites of the non-unique primer where the differential extension has not made the primer sufficiently stable to prime. However, the primer extended at the selected stretch is sufficiently stable to prime.

  8. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Winston Chen, C.H.; Taranenko, N.I.; Zhu, Y.F.; Chung, C.N.; Allman, S.L.

    1997-03-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, the authors recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Snager`s enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. The preliminary results indicate laser mass spectrometry can possibly be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, the authors applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  9. Elucidating population histories using genomic DNA sequences.

    Science.gov (United States)

    Vigilant, Linda

    2009-04-01

    In 1993, Cliff Jolly suggested that rather than debating species definitions and classifications, energy would be better spent investigating multidimensional patterns of variation and gene flow among populations. Until now, however, genetic studies of wild primate populations have been limited to very small portions of the genome. Access to complete genome sequences of humans, chimpanzees, macaques, and other primates makes it possible to design studies surveying substantial amounts of DNA sequence variation at multiple genetic loci in representatives of closely related but distinct wild primate populations. Such data can be analyzed with new approaches that estimate not only when populations diverged but also the relative amounts and directions of subsequent gene flow. These analyses will reemphasize the difficulty of achieving consistent species and subspecies definitions by revealing the extent of variation in the amount and duration of gene flow accompanying population divergences.

  10. DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

    Science.gov (United States)

    Edwards, John R.; Itagaki, Yasuhiro; Ju, Jingyue

    2001-01-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing. PMID:11691941

  11. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  12. Silicene nanoribbon as a new DNA sequencing device

    Science.gov (United States)

    Alesheikh, Sara; Shahtahmassebi, Nasser; Roknabadi, Mahmood Rezaee; Pilevar Shahri, Raheleh

    2018-02-01

    The importance of applying DNA sequencing in different fields, results in looking for fast and cheap methods. Nanotechnology helps this development by introducing nanostructures used for DNA sequencing. In this work we study the interaction between zigzag silicene nanoribbon and DNA nucleobases using DFT and non equilibrium Green's function approach, to investigate the possibility of using zigzag silicene nanoribbons as a biosensor for DNA sequencing.

  13. A synthetic DNA motor that transports nanoparticles along carbon nanotubes

    Science.gov (United States)

    Cha, Tae-Gon; Pan, Jing; Chen, Haorong; Salgado, Janette; Li, Xiang; Mao, Chengde; Choi, Jong Hyun

    2014-01-01

    Intracellular protein motors have evolved to perform specific tasks critical to the function of cells such as intracellular trafficking and cell division. Kinesin and dynein motors, for example, transport cargoes in living cells by walking along microtubules powered by adenosine triphosphate hydrolysis. These motors can make discrete 8 nm centre-of-mass steps and can travel over 1 µm by changing their conformations during the course of adenosine triphosphate binding, hydrolysis and product release. Inspired by such biological machines, synthetic analogues have been developed including self-assembled DNA walkers that can make stepwise movements on RNA/DNA substrates or can function as programmable assembly lines. Here, we show that motors based on RNA-cleaving DNA enzymes can transport nanoparticle cargoes--CdS nanocrystals in this case--along single-walled carbon nanotubes. Our motors extract chemical energy from RNA molecules decorated on the nanotubes and use that energy to fuel autonomous, processive walking through a series of conformational changes along the one-dimensional track. The walking is controllable and adapts to changes in the local environment, which allows us to remotely direct `go' and `stop' actions. The translocation of individual motors can be visualized in real time using the visible fluorescence of the cargo nanoparticle and the near-infared emission of the carbon-nanotube track. We observed unidirectional movements of the molecular motors over 3 µm with a translocation velocity on the order of 1 nm min-1 under our experimental conditions.

  14. Identification of Meconopsis species by a DNA barcode sequence ...

    African Journals Online (AJOL)

    Deoxyribonucleic acid (DNA) barcoding is a novel technology that uses a standard DNA sequence to facilitate species identification. Species identification is necessary for the authentication of traditional plant based medicines. Although a consensus has not been agreed regarding which DNA sequences can be used as ...

  15. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length ...

  16. SWORDS: A statistical tool for analysing large DNA sequences

    Indian Academy of Sciences (India)

    In this article, we present some simple yet effective statistical techniques for analysing and comparing large DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain ...

  17. SWORDS: A statistical tool for analysing large DNA sequences

    Indian Academy of Sciences (India)

    Unknown

    In this article, we present some simple yet effective statistical techniques for analysing and comparing large. DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain ...

  18. Immobilization, hybridization, and oxidation of synthetic DNA on gold surface: Electron transfer investigated by electrochemistry and scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    McEwen, Gerald D.; Chen Fan [Biological Engineering Program, Department of Biological and Irrigation Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105 (United States); Zhou Anhong, E-mail: Anhong.Zhou@usu.edu [Biological Engineering Program, Department of Biological and Irrigation Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105 (United States)

    2009-06-08

    Fundamental understanding of interfacial electron transfer (ET) among electrolyte/DNA/solid-surface will facilitate the design for electrical detection of DNA molecules. In this report, the electron transfer characteristics of synthetic DNA (sequence from pathogenic Cryptosporidium parvum) self-assembled on a gold surface was electrochemically studied. The effects of immobilization order on the interface ET related parameters such as diffusion coefficient (D{sub 0}), surface coverage ({theta}{sub R}), and monolayer thickness (d{sub i}) were determined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). DNA surface density ({Gamma}{sub DNA}) was determined by the integration of the charge of the electro-oxidation current peaks during the initial cyclic voltammetry scans. It was found that the DNA surface densities at different modifications followed the order: {Gamma}{sub DNA} (dsS-DNA/Au) > {Gamma}{sub DNA} (MCH/dsS-DNA/Au) > {Gamma}{sub DNA} (dsS-DNA/MCH/Au). It was also revealed that the electro-oxidation of the DNA modified gold surface would involve the oxidation of nucleotides (guanine and adenine) with a 5.51 electron transfer mechanism and the oxidative desorption of DNA and MCH molecules by a 3 electron transfer mechanism. STM topography and current image analysis indicated that the surface conductivity after each surface modification followed the order: dsS-DNA/Au < MCH/dsS-DNA/Au < oxidized MCH/dsS-DNA/Au < Hoechst/oxidized MCH/dsS-DNA/Au. The results from this study suggested a combination of variations in immobilization order may provide an alternative approach for the optimization of DNA hybridization and the further development for electrical detection of DNA.

  19. Feature Extraction From DNA Sequences by Multifractal Analysis

    National Research Council Canada - National Science Library

    Zhang, H

    2001-01-01

    This paper presents feature extraction and estimation of multifractal measures of DNA sequences using a multifractal methodology and demonstrates a new scheme for identifying biological functionality...

  20. High molecular weight DNA assembly in vivo for synthetic biology applications.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2017-05-01

    DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

  1. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

  2. An automated annotation tool for genomic DNA sequences using

    Indian Academy of Sciences (India)

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

  3. Random Coding Bounds for DNA Codes Based on Fibonacci Ensembles of DNA Sequences

    Science.gov (United States)

    2008-07-01

    Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188) Washington, DC...COVERED (From - To) 6 Jul 08 – 11 Jul 08 4. TITLE AND SUBTITLE RANDOM CODING BOUNDS FOR DNA CODES BASED ON FIBONACCI ENSEMBLES OF DNA SEQUENCES...sequences which are generalizations of the Fibonacci sequences. 15. SUBJECT TERMS DNA Codes, Fibonacci Ensembles, DNA Computing, Code Optimization 16

  4. DNA sequencing by denaturation: principle and thermodynamic simulations.

    Science.gov (United States)

    Chen, Ying-Ja; Huang, Xiaohua

    2009-01-01

    We describe a new DNA sequencing method called sequencing by denaturation (SBD). A Sanger dideoxy sequencing reaction is performed on the templates on a solid surface to generate a ladder of DNA fragments randomly terminated by fluorescently labeled dideoxyribonucleotides. The labeled DNA fragments are sequentially denatured from the templates and the process is monitored by measuring the change in fluorescence intensities from the surface. By analyzing the denaturation profiles, the base sequence of the template can be determined. Using thermodynamic principles, we simulated the denaturation profiles of a series of oligonucleotides ranging from 12 to 32 bases and developed a base-calling algorithm to decode the sequences. These simulations demonstrate that DNA molecules up to 20 bases can be sequenced by SBD. Experimental measurements of the melting profiles of DNA fragments in solution confirm that DNA sequences can be determined by SBD. The potential limitations and advantages of SBD are discussed. With SBD, millions of sequencing reactions can be performed on a small area on a surface in parallel with a very small amount of sequencing reagents. Therefore, DNA sequencing by SBD could potentially result in a significant increase in speed and reduction in cost in large-scale genome resequencing.

  5. Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

    Science.gov (United States)

    Peralta-Castro, Antolín; Baruch-Torres, Noe; Brieba, Luis G

    2017-10-13

    DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5'-(G/C)GGA-3' template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5'-GA-3') as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Nanopores: A journey towards DNA sequencing

    Science.gov (United States)

    Wanunu, Meni

    2013-01-01

    Much more than ever, nucleic acids are recognized as key building blocks in many of life's processes, and the science of studying these molecular wonders at the single-molecule level is thriving. A new method of doing so has been introduced in the mid 1990's. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes. These conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current. In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest. Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics. This review surveys past and current studies related to nucleic acid biophysics, and will hopefully provoke a discussion of immediate and future prospects for the field. PMID:22658507

  7. Levenshtein error-correcting barcodes for multiplexed DNA sequencing

    NARCIS (Netherlands)

    Buschmann, Tilo; Bystrykh, Leonid V.

    2013-01-01

    Background: High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called

  8. Sequence-specific packaging of DNA in human sperm chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Gatewood, J.M.; Cook, G.R.; Balhorn, R.; Bradbury, E.M.; Schmid, C.W.

    1987-05-22

    The DNA in human sperm chromatin is packaged into nucleoprotamine (approx.85%) and nucleohistone (approx.15%). Whether these two chromatin fractions are sequence-specific subsets of the spermatozoon genome is the question addressed in this report. Sequence-specific packaging would suggest distinct structural and functional roles for nucleohistone and nucleoprotamine in late spermatogenesis or early development or both. After removal of histones with 0.65 M NaCl, exposed DNA was cleaved with Bam HI restriction endonuclease and separated by centrifugation from insoluble nucleoprotamine. The DNA sequence distribution of nucleohistone DNA in the supernatant and nucleoprotamine DNA in the pellet was compared by cloning size-selected single-copy sequences and by using the derived clones as probes of nucleohistone DNA and nucleoprotamine DNA. Two clones derived from nucleohistone DNA preferentially hybridized to nucleohistone DNA, and two clones derived from nucleoprotamine DNA preferentially hybridized to nucleoprotamine DNA, which demonstrated the existence of sequence-specific nucleohistone and nucleoprotamine components within the human spermatozoon.

  9. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

    Science.gov (United States)

    Currin, Andrew; Swainston, Neil; Day, Philip J.

    2015-01-01

    The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole

  10. Molecular design of sequence specific DNA alkylating agents.

    Science.gov (United States)

    Minoshima, Masafumi; Bando, Toshikazu; Shinohara, Ken-ichi; Sugiyama, Hiroshi

    2009-01-01

    Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

  11. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis

    Science.gov (United States)

    Chen, C. H. Winston; Taranenko, N. I.; Golovlev, V. V.; Isola, N. R.; Allman, S. L.

    1998-03-01

    Rapid DNA sequencing and/or analysis is critically important for biomedical research. In the past, gel electrophoresis has been the primary tool to achieve DNA analysis and sequencing. However, gel electrophoresis is a time-consuming and labor-extensive process. Recently, we have developed and used laser desorption mass spectrometry (LDMS) to achieve sequencing of ss-DNA longer than 100 nucleotides. With LDMS, we succeeded in sequencing DNA in seconds instead of hours or days required by gel electrophoresis. In addition to sequencing, we also applied LDMS for the detection of DNA probes for hybridization LDMS was also used to detect short tandem repeats for forensic applications. Clinical applications for disease diagnosis such as cystic fibrosis caused by base deletion and point mutation have also been demonstrated. Experimental details will be presented in the meeting. abstract.

  12. Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

    Science.gov (United States)

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

  13. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  14. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

    Science.gov (United States)

    Chen, Cheng-Yao

    2014-01-01

    Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today’s standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies. PMID:25009536

  15. DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

    Directory of Open Access Journals (Sweden)

    Cheng-Yao eChen

    2014-06-01

    Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  16. Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

    Science.gov (United States)

    Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

    2017-11-13

    A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

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

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles

    1997-01-01

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

  18. [Study on factors influencing DNA sequencing by automatic genetic analyzer].

    Science.gov (United States)

    Yan, Shaofei; Wang, Wei; Xu, Jin; Bai, Li; Gan, Xin; Li, Fengqin

    2015-05-01

    To acquire accurate and successful DNA sequencing in a cost-effective way by ABI3500xl automatic genetic analyzer. BigDye was diluted to 8, 16 and 32 times in PCR product sequencing. Three different methods including CENTRI-SEP kit, BigDye cleaning beads and ethanol-NaAc-EDTA were used to purify the sequencing PCR products. The results of DNA sequencing were correct when BigDye was diluted up to 16 times. The misreading of nucleic acid bases was found as BigDye was diluted to 32 times. All three purification methods provided acceptable DNA sequencing results. In terms of method for purification of PCR products, the CENTRI-SEP Kit was the most expensive but time-saving (0.5 h), while ethanol-NaAc-EDTA method was the most economical but time-consuming (2 h). The BigDye cleaning beads method was of a suitable purification time (1 h) but not fit for high-throughput DNA sequencing. BigDye should be diluted up to 16 times in DNA sequencing by ABI3500xl DNA analyzer. Although all three purification methods may promise DNA sequencing results with good quality, it is necessary to choose an appropriate one to keep the balance between time and cost on the basis of the lab condition.

  19. Order and correlations in genomic DNA sequences. The spectral approach

    International Nuclear Information System (INIS)

    Lobzin, Vasilii V; Chechetkin, Vladimir R

    2000-01-01

    The structural analysis of genomic DNA sequences is discussed in the framework of the spectral approach, which is sufficiently universal due to the reciprocal correspondence and mutual complementarity of Fourier transform length scales. The spectral characteristics of random sequences of the same nucleotide composition possess the property of self-averaging for relatively short sequences of length M≥100-300. Comparison with the characteristics of random sequences determines the statistical significance of the structural features observed. Apart from traditional applications to the search for hidden periodicities, spectral methods are also efficient in studying mutual correlations in DNA sequences. By combining spectra for structure factors and correlation functions, not only integral correlations can be estimated but also their origin identified. Using the structural spectral entropy approach, the regularity of a sequence can be quantitatively assessed. A brief introduction to the problem is also presented and other major methods of DNA sequence analysis described. (reviews of topical problems)

  20. Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation

    Energy Technology Data Exchange (ETDEWEB)

    Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R.; Carrano, A.

    1995-01-25

    Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.

  1. An auditory display tool for DNA sequence analysis.

    Science.gov (United States)

    Temple, Mark D

    2017-04-24

    DNA Sonification refers to the use of an auditory display to convey the information content of DNA sequence data. Six sonification algorithms are presented that each produce an auditory display. These algorithms are logically designed from the simple through to the more complex. Three of these parse individual nucleotides, nucleotide pairs or codons into musical notes to give rise to 4, 16 or 64 notes, respectively. Codons may also be parsed degenerately into 20 notes with respect to the genetic code. Lastly nucleotide pairs can be parsed as two separate frames or codons can be parsed as three reading frames giving rise to multiple streams of audio. The most informative sonification algorithm reads the DNA sequence as codons in three reading frames to produce three concurrent streams of audio in an auditory display. This approach is advantageous since start and stop codons in either frame have a direct affect to start or stop the audio in that frame, leaving the other frames unaffected. Using these methods, DNA sequences such as open reading frames or repetitive DNA sequences can be distinguished from one another. These sonification tools are available through a webpage interface in which an input DNA sequence can be processed in real time to produce an auditory display playable directly within the browser. The potential of this approach as an analytical tool is discussed with reference to auditory displays derived from test sequences including simple nucleotide sequences, repetitive DNA sequences and coding or non-coding genes. This study presents a proof-of-concept that some properties of a DNA sequence can be identified through sonification alone and argues for their inclusion within the toolkit of DNA sequence browsers as an adjunct to existing visual and analytical tools.

  2. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Unknown

    Introduction. DNA sequencing has evolved from a complicated labo- ratory process to an automated technique using high- throughput sequencers with fluorescent-dye-based chemistry. This technological advance coupled with the replacement of the traditional mapping and sequencing of clones in series to an integrated ...

  3. Illumina Sequencing of Bisulfite-Converted DNA Libraries.

    Science.gov (United States)

    Lizardi, Paul M; Yan, Qin; Wajapeyee, Narendra

    2017-11-01

    Here we describe a standard MethylC-seq protocol using single-read sequencing on an Illumina Genome Analyzer II platform. The protocol involves ligation of methylated sequencing adaptors to sonicated genomic DNA, gel purification, sodium bisulfite conversion, polymerase chain reaction (PCR) amplification, and sequencing. © 2017 Cold Spring Harbor Laboratory Press.

  4. Simulations Using Random-Generated DNA and RNA Sequences

    Science.gov (United States)

    Bryce, C. F. A.

    1977-01-01

    Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

  5. Purification of sequence-specific DNA-binding proteins by affinity chromatography.

    Science.gov (United States)

    Kerrigan, L A; Kadonaga, J T

    2001-05-01

    Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence-specific DNA-binding properties. The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr-activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

  6. A mathematical model and numerical method for thermoelectric DNA sequencing

    Science.gov (United States)

    Shi, Liwei; Guilbeau, Eric J.; Nestorova, Gergana; Dai, Weizhong

    2014-05-01

    Single nucleotide polymorphisms (SNPs) are single base pair variations within the genome that are important indicators of genetic predisposition towards specific diseases. This study explores the feasibility of SNP detection using a thermoelectric sequencing method that measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a DNA strand. We propose a three-dimensional mathematical model that governs the DNA sequencing device with a reaction zone that contains DNA template/primer complex immobilized to the surface of the lower channel wall. The model is then solved numerically. Concentrations of reactants and the temperature distribution are obtained. Results indicate that when the nucleoside is complementary to the next base in the DNA template, polymerization occurs lengthening the complementary polymer and releasing thermal energy with a measurable temperature change, implying that the thermoelectric conceptual device for sequencing DNA may be feasible for identifying specific genes in individuals.

  7. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    International Nuclear Information System (INIS)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S.; Arbuthnot, Patrick

    2009-01-01

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  8. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  9. Laser desorption mass spectrometry for DNA analysis and sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.H.; Taranenko, N.I.; Tang, K.; Allman, S.L.

    1995-03-01

    Laser desorption mass spectrometry has been considered as a potential new method for fast DNA sequencing. Our approach is to use matrix-assisted laser desorption to produce parent ions of DNA segments and a time-of-flight mass spectrometer to identify the sizes of DNA segments. Thus, the approach is similar to gel electrophoresis sequencing using Sanger`s enzymatic method. However, gel, radioactive tagging, and dye labeling are not required. In addition, the sequencing process can possibly be finished within a few hundred microseconds instead of hours and days. In order to use mass spectrometry for fast DNA sequencing, the following three criteria need to be satisfied. They are (1) detection of large DNA segments, (2) sensitivity reaching the femtomole region, and (3) mass resolution good enough to separate DNA segments of a single nucleotide difference. It has been very difficult to detect large DNA segments by mass spectrometry before due to the fragile chemical properties of DNA and low detection sensitivity of DNA ions. We discovered several new matrices to increase the production of DNA ions. By innovative design of a mass spectrometer, we can increase the ion energy up to 45 KeV to enhance the detection sensitivity. Recently, we succeeded in detecting a DNA segment with 500 nucleotides. The sensitivity was 100 femtomole. Thus, we have fulfilled two key criteria for using mass spectrometry for fast DNA sequencing. The major effort in the near future is to improve the resolution. Different approaches are being pursued. When high resolution of mass spectrometry can be achieved and automation of sample preparation is developed, the sequencing speed to reach 500 megabases per year can be feasible.

  10. 50 CFR 23.16 - What are the U.S. CITES requirements for urine, feces, and synthetically derived DNA?

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false What are the U.S. CITES requirements for... Requirements § 23.16 What are the U.S. CITES requirements for urine, feces, and synthetically derived DNA? (a...) DNA. We differentiate between DNA directly extracted from blood and tissue and DNA synthetically...

  11. An Optimal Seed Based Compression Algorithm for DNA Sequences

    Directory of Open Access Journals (Sweden)

    Pamela Vinitha Eric

    2016-01-01

    Full Text Available This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms.

  12. Statistical assignment of DNA sequences using Bayesian phylogenetics

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Huelsenbeck, John P.

    2008-01-01

    We provide a new automated statistical method for DNA barcoding based on a Bayesian phylogenetic analysis. The method is based on automated database sequence retrieval, alignment, and phylogenetic analysis using a custom-built program for Bayesian phylogenetic analysis. We show on real data......-analysis of previously published ancient DNA data and show that, with high statistical confidence, most of the published sequences are in fact of Neanderthal origin. However, there are several cases of chimeric sequences that are comprised of a combination of both Neanderthal and modern human DNA....

  13. An Optimal Seed Based Compression Algorithm for DNA Sequences.

    Science.gov (United States)

    Eric, Pamela Vinitha; Gopalakrishnan, Gopakumar; Karunakaran, Muralikrishnan

    2016-01-01

    This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms.

  14. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    F counterselection was developed to directly select for compounds able to disrupt selected interactions. We have subsequently constructed a cyclic peptide library for intracellular synthesis of cyclic peptides using known technology. Several cyclic peptides were able to interfere with oligomerization of Dna......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...... effect of the peptides confirming the target of the peptides....

  15. Characteristics of alternating current hopping conductivity in DNA sequences

    International Nuclear Information System (INIS)

    Song-Shan, Ma; Hui, Xu; Huan-You, Wang; Rui, Guo

    2009-01-01

    This paper presents a model to describe alternating current (AC) conductivity of DNA sequences, in which DNA is considered as a one-dimensional (1D) disordered system, and electrons transport via hopping between localized states. It finds that AC conductivity in DNA sequences increases as the frequency of the external electric field rises, and it takes the form of ø ac (ω) ∼ ω 2 ln 2 (1/ω). Also AC conductivity of DNA sequences increases with the increase of temperature, this phenomenon presents characteristics of weak temperature-dependence. Meanwhile, the AC conductivity in an off-diagonally correlated case is much larger than that in the uncorrelated case of the Anderson limit in low temperatures, which indicates that the off-diagonal correlations in DNA sequences have a great effect on the AC conductivity, while at high temperature the off-diagonal correlations no longer play a vital role in electric transport. In addition, the proportion of nucleotide pairs p also plays an important role in AC electron transport of DNA sequences. For p < 0.5, the conductivity of DNA sequence decreases with the increase of p, while for p ≥ 0.5, the conductivity increases with the increase of p. (cross-disciplinary physics and related areas of science and technology)

  16. Plasmonic Nanopores for Trapping, Controlling Displacement, and Sequencing of DNA.

    Science.gov (United States)

    Belkin, Maxim; Chao, Shu-Han; Jonsson, Magnus P; Dekker, Cees; Aksimentiev, Aleksei

    2015-11-24

    With the aim of developing a DNA sequencing methodology, we theoretically examine the feasibility of using nanoplasmonics to control the translocation of a DNA molecule through a solid-state nanopore and to read off sequence information using surface-enhanced Raman spectroscopy. Using molecular dynamics simulations, we show that high-intensity optical hot spots produced by a metallic nanostructure can arrest DNA translocation through a solid-state nanopore, thus providing a physical knob for controlling the DNA speed. Switching the plasmonic field on and off can displace the DNA molecule in discrete steps, sequentially exposing neighboring fragments of a DNA molecule to the pore as well as to the plasmonic hot spot. Surface-enhanced Raman scattering from the exposed DNA fragments contains information about their nucleotide composition, possibly allowing the identification of the nucleotide sequence of a DNA molecule transported through the hot spot. The principles of plasmonic nanopore sequencing can be extended to detection of DNA modifications and RNA characterization.

  17. Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

    International Nuclear Information System (INIS)

    Steenbergh, P.H.

    1979-01-01

    The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

  18. A synthetic DNA and fusion PCR approach to the ectopic expression of high levels of the D1 protein of photosystem II in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Nagarajan, Aparna; Winter, Regan; Eaton-Rye, Julian; Burnap, Robert

    2011-01-01

    A hybrid approach involving synthetic DNA, fusion PCR, and ectopic expression has been used to genetically manipulate the expression of the D1 protein of photosystem II (PSII) in the model cyanobacterium Synechocystis sp. PCC6803. Due to the toxicity of the full-length psbA gene in E. coli, a chimeric psbA2 gene locus was commercially synthesised and cloned in two halves. High-fidelity fusion PCR utilizing sequence overlap between the two synthetic gene halves allowed the production of a DNA fragment that was able to recombine the full-length psbA2 gene into the Synechocystis chromosome at an ectopic (non-native) location. This was accomplished by designing the synthetic DNA/fusion PCR product to have the psbA2 gene, with control sequences, interposed between chimeric sequences corresponding to an ectopic target chromosomal location. Additionally, a recipient strain of Synechocystis lacking all three psbA genes was produced by a combination of traditional marker replacement and markerless replacement techniques. Transformation of this multiple deletion strain by the synthetic DNA/fusion PCR product faithfully restored D1 expression in terms of its expression and PSII repair capacity. The advantages and potential issues for using this approach to rapidly introduce chimeric sequence characteristics as a general tool to produce novel genetic constructs are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. ATRF Houses the Latest DNA Sequencing Technologies | Poster

    Science.gov (United States)

    By Ashley DeVine, Staff Writer By the end of October, the Advanced Technology Research Facility (ATRF) will be one of the few facilities in the world to house all of the latest DNA sequencing technologies.

  20. DNA sequencing using polymerase substrate-binding kinetics.

    Science.gov (United States)

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-23

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.

  1. Levenshtein error-correcting barcodes for multiplexed DNA sequencing.

    Science.gov (United States)

    Buschmann, Tilo; Bystrykh, Leonid V

    2013-09-11

    High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag or barcode that is attached to the sequencing or amplification primer and hence appears at the beginning of the sequence in every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and Levenshtein codes. Levenshtein codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this paper we demonstrate the decreased error correction capability of Levenshtein codes in a DNA context and suggest an adaptation of Levenshtein codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaption we take the DNA context into account and redefine the word length whenever an insertion or deletion is revealed. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors. We present an adaptation of Levenshtein codes to DNA contexts capable of correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable

  2. Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

    OpenAIRE

    Swerdlow, H; Gesteland, R

    1990-01-01

    Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequenci...

  3. Sigma: multiple alignment of weakly-conserved non-coding DNA sequence

    Directory of Open Access Journals (Sweden)

    Siddharthan Rahul

    2006-03-01

    Full Text Available Abstract Background Existing tools for multiple-sequence alignment focus on aligning protein sequence or protein-coding DNA sequence, and are often based on extensions to Needleman-Wunsch-like pairwise alignment methods. We introduce a new tool, Sigma, with a new algorithm and scoring scheme designed specifically for non-coding DNA sequence. This problem acquires importance with the increasing number of published sequences of closely-related species. In particular, studies of gene regulation seek to take advantage of comparative genomics, and recent algorithms for finding regulatory sites in phylogenetically-related intergenic sequence require alignment as a preprocessing step. Much can also be learned about evolution from intergenic DNA, which tends to evolve faster than coding DNA. Sigma uses a strategy of seeking the best possible gapless local alignments (a strategy earlier used by DiAlign, at each step making the best possible alignment consistent with existing alignments, and scores the significance of the alignment based on the lengths of the aligned fragments and a background model which may be supplied or estimated from an auxiliary file of intergenic DNA. Results Comparative tests of sigma with five earlier algorithms on synthetic data generated to mimic real data show excellent performance, with Sigma balancing high "sensitivity" (more bases aligned with effective filtering of "incorrect" alignments. With real data, while "correctness" can't be directly quantified for the alignment, running the PhyloGibbs motif finder on pre-aligned sequence suggests that Sigma's alignments are superior. Conclusion By taking into account the peculiarities of non-coding DNA, Sigma fills a gap in the toolbox of bioinformatics.

  4. An extended sequence specificity for UV-induced DNA damage.

    Science.gov (United States)

    Chung, Long H; Murray, Vincent

    2018-01-01

    The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  5. Novel Graphical Representation and Numerical Characterization of DNA Sequences

    Directory of Open Access Journals (Sweden)

    Chun Li

    2016-02-01

    Full Text Available Modern sequencing technique has provided a wealth of data on DNA sequences, which has made the analysis and comparison of sequences a very important but difficult task. In this paper, by regarding the dinucleotide as a 2-combination of the multiset { ∞ · A , ∞ · G , ∞ · C , ∞ · T } , a novel 3-D graphical representation of a DNA sequence is proposed, and its projections on planes (x,y, (y,z and (x,z are also discussed. In addition, based on the idea of “piecewise function”, a cell-based descriptor vector is constructed to numerically characterize the DNA sequence. The utility of our approach is illustrated by the examination of phylogenetic analysis on four datasets.

  6. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  7. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing

    NARCIS (Netherlands)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B. M.; Cornel, Martina C.; Sistermans, Erik A.

    2016-01-01

    Cell-free DNA (cf DNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide

  8. DNA Sequences of RAPD Fragments in the Egyptian cotton ...

    African Journals Online (AJOL)

    Random Amplified Polymorphic DNAs (RAPDs) is a DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. Despite the fact that the RAPD technique has become a very powerful tool and has found use in numerous applications, yet, the nature of ...

  9. Effects of sequence on DNA wrapping around histones

    Science.gov (United States)

    Ortiz, Vanessa

    2011-03-01

    A central question in biophysics is whether the sequence of a DNA strand affects its mechanical properties. In epigenetics, these are thought to influence nucleosome positioning and gene expression. Theoretical and experimental attempts to answer this question have been hindered by an inability to directly resolve DNA structure and dynamics at the base-pair level. In our previous studies we used a detailed model of DNA to measure the effects of sequence on the stability of naked DNA under bending. Sequence was shown to influence DNA's ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. Here, we have now included histone-DNA interactions to see if the results obtained for naked DNA are transferable to the problem of nucleosome positioning. Different DNA sequences interacting with the histone protein complex are studied, and their equilibrium and mechanical properties are compared among themselves and with the naked case. NLM training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM T15LM007359).

  10. Googling DNA sequences on the World Wide Web.

    Science.gov (United States)

    Hajibabaei, Mehrdad; Singer, Gregory A C

    2009-11-10

    New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

  11. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    Science.gov (United States)

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  12. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    DEFF Research Database (Denmark)

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie

    2014-01-01

    sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5'-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections...

  13. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Unknown

    , New Delhi 110 067, India. Abstract ... analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by .... genes for the TCA cycle, while in mitochondria only a subset of the ...

  14. Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences

    DEFF Research Database (Denmark)

    Svitashev, S.; Bryngelsson, T.; Vershinin, A.

    1994-01-01

    A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. In situ hybridization experiments showed dispersed organization of the sequences...

  15. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...

  16. An integer programming approach to DNA sequence assembly.

    Science.gov (United States)

    Chang, Youngjung; Sahinidis, Nikolaos V

    2011-08-10

    De novo sequence assembly is a ubiquitous combinatorial problem in all DNA sequencing technologies. In the presence of errors in the experimental data, the assembly problem is computationally challenging, and its solution may not lead to a unique reconstruct. The enumeration of all alternative solutions is important in drawing a reliable conclusion on the target sequence, and is often overlooked in the heuristic approaches that are currently available. In this paper, we develop an integer programming formulation and global optimization solution strategy to solve the sequence assembly problem with errors in the data. We also propose an efficient technique to identify all alternative reconstructs. When applied to examples of sequencing-by-hybridization, our approach dramatically increases the length of DNA sequences that can be handled with global optimality certificate to over 10,000, which is more than 10 times longer than previously reported. For some problem instances, alternative solutions exhibited a wide range of different ability in reproducing the target DNA sequence. Therefore, it is important to utilize the methodology proposed in this paper in order to obtain all alternative solutions to reliably infer the true reconstruct. These alternative solutions can be used to refine the obtained results and guide the design of further experiments to correctly reconstruct the target DNA sequence. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

    Science.gov (United States)

    Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

    2017-11-14

    Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

  18. An artificial intelligence approach to DNA sequence feature recognition.

    Science.gov (United States)

    Mural, R J; Einstein, J R; Guan, X; Mann, R C; Uberbacher, E C

    1992-01-01

    The ultimate goal of the Human Genome project is to extract the biologically relevant information recorded in the estimated 100,000 genes encoded by the 3 x 10(9) bases of the human genome. This necessitates development of reliable computer-based methods capable of analysing and correctly identifying genes in the vast amounts of DNA-sequence data generated. Such tools may save time and labour by simplifying, for example, screening of cDNA libraries. They may also facilitate the localization of human disease genes by identifying candidate genes in promising regions of anonymous DNA sequence.

  19. Mitochondrial DNA sequence-based phylogenetic relationship ...

    Indian Academy of Sciences (India)

    The phylogenetic relationships among flesh flies of the family Sarcophagidae has been based mainly on the morphology of male genitalia. However, the male genitalic character-based relationships are far from satisfactory. Therefore, in the present study mitochondrial DNA has been used as marker to unravel genetic ...

  20. Mitochondrial DNA sequence-based phylogenetic relationship ...

    Indian Academy of Sciences (India)

    2007 Population structure of the malaria vector Anopheles dar- lingi in Rondonia, Brazilian Amazon, based on mitochondrial. DNA. Mem. Inst. Oswaldo Cruz 102, 953–958. Avise J. C. 2004 Molecular markers, natural history, and evolution,. 2nd edition. Sinauer, Sunderland, USA. Cameron S. L., Lambkin C. L., Barker S. C. ...

  1. Plant viral intergenic DNA sequence repeats with transcription enhancing activity

    Directory of Open Access Journals (Sweden)

    Cazzonelli Christopher I

    2005-02-01

    Full Text Available Abstract Background The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection. Results DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p Conclusion Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE, and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.

  2. Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

    Directory of Open Access Journals (Sweden)

    Jian eWu

    2012-11-01

    Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

  3. Sequence-selective single-molecule alkylation with a pyrrole-imidazole polyamide visualized in a DNA nanoscaffold.

    Science.gov (United States)

    Yoshidome, Tomofumi; Endo, Masayuki; Kashiwazaki, Gengo; Hidaka, Kumi; Bando, Toshikazu; Sugiyama, Hiroshi

    2012-03-14

    We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.

  4. Mapping Base Modifications in DNA by Transverse-Current Sequencing

    Science.gov (United States)

    Alvarez, Jose R.; Skachkov, Dmitry; Massey, Steven E.; Kalitsov, Alan; Velev, Julian P.

    2018-02-01

    Sequencing DNA modifications and lesions, such as methylation of cytosine and oxidation of guanine, is even more important and challenging than sequencing the genome itself. The traditional methods for detecting DNA modifications are either insensitive to these modifications or require additional processing steps to identify a particular type of modification. Transverse-current sequencing in nanopores can potentially identify the canonical bases and base modifications in the same run. In this work, we demonstrate that the most common DNA epigenetic modifications and lesions can be detected with any predefined accuracy based on their tunneling current signature. Our results are based on simulations of the nanopore tunneling current through DNA molecules, calculated using nonequilibrium electron-transport methodology within an effective multiorbital model derived from first-principles calculations, followed by a base-calling algorithm accounting for neighbor current-current correlations. This methodology can be integrated with existing experimental techniques to improve base-calling fidelity.

  5. Next-generation sequencing offers new insights into DNA degradation

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Orlando, Ludovic Antoine Alexandre; Willerslev, Eske

    2012-01-01

    The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation...... rates that previously were obtained only from extrapolations of results from in vitro kinetic experiments performed over short timescales. For example, recent next-generation sequencing of ancient DNA reveals purine bases as one of the main targets of postmortem hydrolytic damage, through base...... elimination and strand breakage. It also shows substantially increased rates of DNA base-loss at guanosine. In this review, we argue that the latter results from an electron resonance structure unique to guanosine rather than adenosine having an extra resonance structure over guanosine as previously suggested....

  6. Sequence-Dependent Persistence Length of Long DNA

    Science.gov (United States)

    Chuang, Hui-Min; Reifenberger, Jeffrey G.; Cao, Han; Dorfman, Kevin D.

    2017-12-01

    Using a high-throughput genome-mapping approach, we obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm ×41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5-393 kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk's theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.

  7. DNA Sequence Analysis in Clinical Medicine, Proceeding Cautiously

    Directory of Open Access Journals (Sweden)

    Moyra Smith

    2017-05-01

    Full Text Available Delineation of underlying genomic and genetic factors in a specific disease may be valuable in establishing a definitive diagnosis and may guide patient management and counseling. In addition, genetic information may be useful in identification of at risk family members. Gene mapping and initial genome sequencing data enabled the development of microarrays to analyze genomic variants. The goal of this review is to consider different generations of sequencing techniques and their application to exome sequencing and whole genome sequencing and their clinical applications. In recent decades, exome sequencing has primarily been used in patient studies. Discussed in some detail, are important measures that have been developed to standardize variant calling and to assess pathogenicity of variants. Examples of cases where exome sequencing has facilitated diagnosis and led to improved medical management are presented. Whole genome sequencing and its clinical relevance are presented particularly in the context of analysis of nucleotide and structural genomic variants in large population studies and in certain patient cohorts. Applications involving analysis of cell free DNA in maternal blood for prenatal diagnosis of specific autosomal trisomies are reviewed. Applications of DNA sequencing to diagnosis and therapeutics of cancer are presented. Also discussed are important recent diagnostic applications of DNA sequencing in cancer, including analysis of tumor derived cell free DNA and exosomes that are present in body fluids. Insights gained into underlying pathogenetic mechanisms of certain complex common diseases, including schizophrenia, macular degeneration, neurodegenerative disease are presented. The relevance of different types of variants, rare, uncommon, and common to disease pathogenesis, and the continuum of causality, are addressed. Pharmogenetic variants detected by DNA sequence analysis are gaining in importance and are particularly relevant

  8. Directed PCR-free engineering of highly repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Preissler Steffen

    2011-09-01

    Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

  9. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    Science.gov (United States)

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing.

    Science.gov (United States)

    Guo, Yaqiong; Li, Na; Lysén, Colleen; Frace, Michael; Tang, Kevin; Sammons, Scott; Roellig, Dawn M; Feng, Yaoyu; Xiao, Lihua

    2015-02-01

    Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences.

    Directory of Open Access Journals (Sweden)

    Ryuji J Machida

    Full Text Available BACKGROUND: Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. METHODOLOGY/PRINCIPAL FINDINGS: A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. CONCLUSIONS/SIGNIFICANCE: Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans.

  12. Mitochondrial DNA sequence variation in Drosophilid species ...

    Indian Academy of Sciences (India)

    Here, we assessed genetic variations in three mitochondrial genes, namely, 16S rRNA, cytochrome c oxidase subunit I and cytochrome c oxidase subunit II (COI and COII) in 26 drosophilid species collected along altitudinal transect from 550 to 2700 m above mean sea level. In the present study, overall 543 sequences ...

  13. A new program for DNA sequence mining

    Indian Academy of Sciences (India)

    Unknown

    activity of proteins by altering their structure (Klintschar and Wiegand 2003). Expressed Sequence Tags ..... among the organisms (for instance; animal versus plant, trees versus annual crops), among the organs (for instance; ... Int. 3rd Balkan Symposium on vegetables and potatoes. Bursa, Turkey, Acta Horticulturae (in ...

  14. The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

    Science.gov (United States)

    Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

    2017-08-21

    Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Improvement of Synthetic Biology Tools for DNA Editing

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda

    The unpredictability and complexity of biological systems limit the development of economically efficient bio-based production processes that rely on renewable carbon sources and are essential for biosustainability and environmental protection. Synthetic biology (synbio) aims at making biology...... easier to engineer and addresses these challenges.The ability to systematically construct, modify and tune biological systems from fully characterized biological components, or parts, is crucial to the success of synbio projects. This thesis aims at contributing to standardization and part sharing...

  16. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    Science.gov (United States)

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.

  17. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein......-protein interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  18. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities.

    Science.gov (United States)

    Troshin, Peter V; Postis, Vincent Lg; Ashworth, Denise; Baldwin, Stephen A; McPherson, Michael J; Barton, Geoffrey J

    2011-03-07

    Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS) that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

  19. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities

    Directory of Open Access Journals (Sweden)

    Baldwin Stephen A

    2011-03-01

    Full Text Available Abstract Background Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

  20. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  1. BioWires: Conductive DNA Nanowires in a Computationally-Optimized, Synthetic Biological Platform for Nanoelectronic Fabrication

    Science.gov (United States)

    Vecchioni, Simon; Toomey, Emily; Capece, Mark C.; Rothschild, Lynn; Wind, Shalom

    2017-01-01

    DNA is an ideal template for a biological nanowire-it has a linear structure several atoms thick; it possesses addressable nucleobase geometry that can be precisely defined; and it is massively scalable into branched networks. Until now, the drawback of DNA as a conducting nanowire been, simply put, its low conductance. To address this deficiency, we extensively characterize a chemical variant of canonical DNA that exploits the affinity of natural cytosine bases for silver ions. We successfully construct chains of single silver ions inside double-stranded DNA, confirm the basic dC-Ag+-dC bond geometry and kinetics, and show length-tunability dependent on mismatch distribution, ion availability and enzyme activity. An analysis of the absorbance spectra of natural DNA and silver-binding, poly-cytosine DNA demonstrates the heightened thermostability of the ion chain and its resistance to aqueous stresses such as precipitation, dialysis and forced reduction. These chemically critical traits lend themselves to an increase in electrical conductivity of over an order of magnitude for 11-base silver-paired duplexes over natural strands when assayed by STM break junction. We further construct and implement a genetic pathway in the E. coli bacterium for the biosynthesis of highly ionizable DNA sequences. Toward future circuits, we construct a model of transcription network architectures to determine the most efficient and robust connectivity for cell-based fabrication, and we perform sequence optimization with a genetic algorithm to identify oligonucleotides robust to changes in the base-pairing energy landscape. We propose that this system will serve as a synthetic biological fabrication platform for more complex DNA nanotechnology and nanoelectronics with applications to deep space and low resource environments.

  2. Β-defensin in Nile tilapia (Oreochromis niloticus): Sequence, tissue expression, and anti-bacterial activity of synthetic peptides.

    Science.gov (United States)

    Dong, Jun-Jian; Wu, Fang; Ye, Xing; Sun, Cheng-Fei; Tian, Yuan-Yuan; Lu, Mai-Xin; Zhang, Rui; Chen, Zhi-Hang

    2015-07-15

    Beta-defensins (β-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the β-defensin (Onβ-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onβ-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onβ-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onβ-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with β-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onβ-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onβ-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onβ-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onβ-defensin plays a role in immune responses that suppress or kill pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Noninvasive prenatal paternity testing (NIPAT) through maternal plasma DNA sequencing

    DEFF Research Database (Denmark)

    Jiang, Haojun; Xie, Yifan; Li, Xuchao

    2016-01-01

    developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels...... in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal...

  4. Dialects of the DNA Uptake Sequence in Neisseriaceae

    Science.gov (United States)

    Frye, Stephan A.; Nilsen, Mariann; Tønjum, Tone; Ambur, Ole Herman

    2013-01-01

    In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation

  5. Dialects of the DNA uptake sequence in Neisseriaceae.

    Directory of Open Access Journals (Sweden)

    Stephan A Frye

    2013-04-01

    Full Text Available In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS, which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS-dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5'-CTG-3' is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS-dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic

  6. Mitochondrial DNA sequence of Onychostoma rara.

    Science.gov (United States)

    Zeng, Chun-Fang; Li, Xiao-Ling; Li, Chuan-Wu; Huang, Xiang-Rong; Wan, Yi-Wen

    2015-01-01

    The complete mitochondrial genome sequence of Onychostoma rara was determined to be 16,590 bp in length and contains 13 protein-coding genes (PCGs), 22 tRNA genes, large (rrnL) and small (rrnS) rRNA and the non-coding control region. Its total A + T content is 55.65%. We also analyzed the structure of control region, 6 CSBs (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F) and 2 bp tandem repeat were detected.

  7. Accelerating Computation of DNA Sequence Alignment in Distributed Environment

    Science.gov (United States)

    Guo, Tao; Li, Guiyang; Deaton, Russel

    Sequence similarity and alignment are most important operations in computational biology. However, analyzing large sets of DNA sequence seems to be impractical on a regular PC. Using multiple threads with JavaParty mechanism, this project has successfully implemented in extending the capabilities of regular Java to a distributed environment for simulation of DNA computation. With the aid of JavaParty and the design of multiple threads, the results of this study demonstrated that the modified regular Java program could perform parallel computing without using RMI or socket communication. In this paper, an efficient method for modeling and comparing DNA sequences with dynamic programming and JavaParty was firstly proposed. Additionally, results of this method in distributed environment have been discussed.

  8. Continuous flow thermal cycler microchip for DNA cycle sequencing.

    Science.gov (United States)

    Wang, Hong; Chen, Jifeng; Zhu, Li; Shadpour, Hamed; Hupert, Mateusz L; Soper, Steven A

    2006-09-01

    We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 degrees C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from benchtop instruments but did not require manual manipulation of the cycle sequencing reactions following amplification.

  9. Multilevel description of the DNA molecule translocation in solid-state synthetic nanopores

    Energy Technology Data Exchange (ETDEWEB)

    Nosik, V. L., E-mail: v-nosik@yandex.ru; Rudakova, E. B. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2016-07-15

    Interest of researchers in micro- and nanofluidics of polymer solutions and, in particular, DNA ionic solutions is constantly increasing. The use of DNA translocation with a controlled velocity through solid-state nanopores and pulsed X-ray beams in new sequencing schemes opens up new possibilities for studying the structure of DNA and other biopolymers. The problems related to the description of DNA molecular motion in a limited volume of nanopore are considered.

  10. Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

    Directory of Open Access Journals (Sweden)

    Thamsborg Stig M

    2009-12-01

    Full Text Available Abstract Background The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing. Methods 31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing. Results Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as A. phagocytophilum by DNA sequencing. Conclusions A. phagocytophilum was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006.

  11. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes......) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6...... relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only...

  12. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

  13. DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  14. An efficient method for generation and subcloning of tandemly repeated DNA sequences with defined length, orientation and spacing.

    Science.gov (United States)

    Jiang, S W; Trujillo, M A; Eberhardt, N L

    1996-08-15

    Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.

  15. Non-radioactive chemical sequencing of biotin labelled DNA.

    OpenAIRE

    Richterich, P

    1989-01-01

    Methods for the nonradioactive chemical sequencing of DNA are described. A biotin marker molecule, attached chemically to an oligonucleotide primer or enzymatically in an endfilling reaction of restriction enzyme sites, is stable during the base-specific chemical modification and strand scission reactions. Following fragment separation by direct blotting electrophoresis, the membrane bound sequence pattern can be visualized by a streptavidin-bridged enzymatic color reaction. The biotin labeli...

  16. Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Ole Herman Ambur

    Full Text Available Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS. In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s of one of the most conserved mechanisms in nature: homologous

  17. Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1

    Directory of Open Access Journals (Sweden)

    Tushar R. Mahajan

    2015-09-01

    Full Text Available The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER, one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

  18. Hydroxylation of deoxyguanosine at 5' site of GG and GGG sequences in double-stranded DNA induced by carbamoyl radicals.

    Science.gov (United States)

    Midorikawa, Kaoru; Hirakawa, Kazutaka; Kawanishi, Shosuke

    2002-06-01

    Free radicals generated by chemicals can cause sequence-specific DNA damage and play important roles in mutagenesis and carcinogenesis. Carbamoyl group (CONH2) and its derived groups (CONR2) occur as natural products and synthetic chemical compounds. We have investigated the DNA damage by carbamoyl radicals .(CONH2), one of carbon-centered radicals. Electron spin resonance (ESR) spectroscopic study has demonstrated that carbamoyl radicals were generated from formamide by treatment with H2O2 plus Cu(II), and from azodicarbonamide by treatment with Cu(II). We have investigated sequence specificity of DNA damage induced by carbamoyl radicals using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 and p53 genes. Treatment of double-stranded DNA with carbamoyl radicals induced an alteration of guanine residues, and subsequent treatment with piperidine or Fpg protein led to chain cleavages at 5'-G of GG and GGG sequences. Carbamoyl radicals enhanced Cu(II)/H2O2-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in double-stranded DNA more efficiently than that in single-stranded DNA. These results shows that carbamoyl radicals specifically induced hydroxylation of deoxyguanosine at 5' site of GG and GGG sequences in double-stranded DNA.

  19. Development of a methodology for the application of synthetic DNA in stream tracer injection experiments

    NARCIS (Netherlands)

    Foppen, J.W.; Seopa, J.; Bakobie, N.; Bogaard, T.

    2013-01-01

    Stream tracer injection experiments are useful for characterizing hydrological and biogeochemical processes in streams. We used nonconservative synthetic DNA and conservative NaCl in six instantaneous tracer injection experiments in streams in the Benelux. The main aim was to compare the performance

  20. Efficient Nuclear DNA Cleavage in Human Cancer Cells by Synthetic Bleomycin Mimics

    NARCIS (Netherlands)

    Li, Qian; van der Wijst, Monique G. P.; Kazemier, Hinke G.; Rots, Marianne G.; Roelfes, Gerard

    Iron complexes of N,N-bis(2-Pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity

  1. RNA-DNA sequence differences spell genetic code ambiguities

    DEFF Research Database (Denmark)

    Bentin, Thomas; Nielsen, Michael L

    2013-01-01

    A recent paper in Science by Li et al. 2011(1) reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. ...

  2. (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 2. Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences. Yan Li Yan Kong Zhe Zhang Yanqiang Yin Bin Liu Guanghui Lv Xiyong Wang. Research Article Volume 93 Issue 2 August 2014 pp 313-323 ...

  3. POSA: perl objects for DNA sequencing data analysis

    NARCIS (Netherlands)

    Aerts, J.A.; Jungerius, B.J.; Groenen, M.A.M.

    2004-01-01

    Background - Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide

  4. POSA : Perl objects for DNA sequencing data analysis

    NARCIS (Netherlands)

    Aerts, JA; Jungerius, BJ; Groenen, MA

    2004-01-01

    Background: Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide

  5. What DNA sequence tells us about gene regulation - The ...

    Indian Academy of Sciences (India)

    Rahul Siddharthan

    2007-11-03

    Nov 3, 2007 ... Predicting cis-regulatory modules: eve enhancers. Performance, even without prior WMs, comparable to dedicated CRM prediction programs like Stubb. Rahul Siddharthan. (The Institute of Mathematical Sciences, Chennai 600 113. What DNA sequence tells us about gene regulation. 03/11/2007. 27 / 34 ...

  6. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  7. DNA sequence and prokaryotic expression analysis of vitellogenin ...

    African Journals Online (AJOL)

    In this study, the DNA sequence of vitellogenin from Antheraea pernyi (Ap-Vg) was identified and its functional domain (30-740 aa, Ap-Vg-1) was expressed in Escherichia coli BL21 (DE3) cells. The recombinant Ap-Vg-1 proteins were purified and used for antibody preparation. The results showed that the intact DNA ...

  8. Mitochondrial DNA sequence variation in the Anatolian Peninsula ...

    Indian Academy of Sciences (India)

    Unknown

    A few studies have previously reported mtDNA sequences in Turks. We attempted to extend these results by analysing a cohort that is not only larger, but also more representative of the. Turkish population living in Anatolia. In order to obtain a descriptive picture for the phylogenetic distribution of the mitochondrial genome ...

  9. Random amplified polymorphic DNA (RAPD) and simple sequence ...

    African Journals Online (AJOL)

    Knowledge as to genetic diversity and relationships among maize hybrids is important for breeding strategies. The main aims of this study were to (1) estimate molecular genetic diversity among 30 maize hybrids by random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers; and (2) compare ...

  10. The white spot syndrome virus DNA genome sequence

    NARCIS (Netherlands)

    Hulten, van M.C.W.; Witteveldt, J.; Peters, S.; Kloosterboer, N.; Tarchini, R.; Fiers, M.; Sandbrink, H.; Klein Lankhorst, R.; Vlak, J.M.

    2001-01-01

    White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6 f the WSSV ORFs

  11. Studies of DNA dumbbells VIII. Melting analysis of DNA dumbbells with dinucleotide repeat stem sequences.

    Science.gov (United States)

    Mandell, Kathleen E; Vallone, Peter M; Owczarzy, Richard; Riccelli, Peter V; Benight, Albert S

    2006-06-15

    Melting curves and circular dichroism spectra were measured for a number of DNA dumbbell and linear molecules containing dinucleotide repeat sequences of different lengths. To study effects of different sequences on the melting and spectroscopic properties, six DNA dumbbells whose stems contain the central sequences (AA)(10), (AC)(10), (AG)(10), (AT)(10), (GC)(10), and (GG)(10) were prepared. These represent the minimal set of 10 possible dinucleotide repeats. To study effects of dinucleotide repeat length, dumbbells with the central sequences (AG)(n), n = 5 and 20, were prepared. Control molecules, dumbbells with a random central sequence, (RN)(n), n = 5, 10, and 20, were also prepared. The central sequence of each dumbbell was flanked on both sides by the same 12 base pairs and T(4) end-loops. Melting curves were measured by optical absorbance and differential scanning calorimetry in solvents containing 25, 55, 85, and 115 mM Na(+). CD spectra were collected from 20 to 45 degrees C and [Na(+)] from 25 to 115 mM. The spectral database did not reveal any apparent temperature dependence in the pretransition region. Analysis of the melting thermodynamics evaluated as a function of Na(+) provided a means for quantitatively estimating the counterion release with melting for the different sequences. Results show a very definite sequence dependence, indicating the salt-dependent properties of duplex DNA are also sequence dependent. Linear DNA molecules containing the (AG)(n) and (RN)(n), sequences, n = 5, 10, 20, and 30, were also prepared and studied. The linear DNA molecules had the exact sequences of the dumbbell stems. That is, the central repeat sequence in each linear duplex was flanked on both sides by the same 12-bp sequence. Melting and CD studies were also performed on the linear DNA molecules. Comparison of results obtained for the same sequences in dumbbell and linear molecular environments reveals several interesting features of the interplay between

  12. Solid-State Nanopore-Based DNA Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Zewen Liu

    2016-01-01

    Full Text Available The solid-state nanopore-based DNA sequencing technology is becoming more and more attractive for its brand new future in gene detection field. The challenges that need to be addressed are diverse: the effective methods to detect base-specific signatures, the control of the nanopore’s size and surface properties, and the modulation of translocation velocity and behavior of the DNA molecules. Among these challenges, the realization of the high-quality nanopores with the help of modern micro/nanofabrication technologies is a crucial one. In this paper, typical technologies applied in the field of solid-state nanopore-based DNA sequencing have been reviewed.

  13. Sequence heterogeneity accelerates protein search for targets on DNA

    International Nuclear Information System (INIS)

    Shvets, Alexey A.; Kolomeisky, Anatoly B.

    2015-01-01

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome

  14. DNA watermarks in non-coding regulatory sequences

    Directory of Open Access Journals (Sweden)

    Pyka Martin

    2009-07-01

    Full Text Available Abstract Background DNA watermarks can be applied to identify the unauthorized use of genetically modified organisms. It has been shown that coding regions can be used to encrypt information into living organisms by using the DNA-Crypt algorithm. Yet, if the sequence of interest presents a non-coding DNA sequence, either the function of a resulting functional RNA molecule or a regulatory sequence, such as a promoter, could be affected. For our studies we used the small cytoplasmic RNA 1 in yeast and the lac promoter region of Escherichia coli. Findings The lac promoter was deactivated by the integrated watermark. In addition, the RNA molecules displayed altered configurations after introducing a watermark, but surprisingly were functionally intact, which has been verified by analyzing the growth characteristics of both wild type and watermarked scR1 transformed yeast cells. In a third approach we introduced a second overlapping watermark into the lac promoter, which did not affect the promoter activity. Conclusion Even though the watermarked RNA and one of the watermarked promoters did not show any significant differences compared to the wild type RNA and wild type promoter region, respectively, it cannot be generalized that other RNA molecules or regulatory sequences behave accordingly. Therefore, we do not recommend integrating watermark sequences into regulatory regions.

  15. Multifractal properties of Hao's geometric representations of DNA sequences

    Science.gov (United States)

    Tiňo, Peter

    2002-02-01

    Hao proposed a graphic representation of subsequence structure in DNA sequences and computed fractal dimensions of such representations for factorizable languages. In this study, we extend Hao's work in several directions: (1) We generalize Hao's scheme to accommodate sequences over an arbitrary finite number of symbols. (2) We establish a direct correspondence between the statistical characterization of symbolic sequences via Rényi entropy spectra and the multifractal characteristics (Rényi generalized dimensions) of the sequences’ spatial representations. (3) We show that for general symbolic dynamical systems, the multifractal fH-spectra in the sequence space endowed with commonly used metrics, coincide with the fH-spectra on Hao's sequence representations. (4) So far the connection between the Hao's scheme and another well-known subsequence visualization scheme-Jeffrey's chaos game representation (CGR)-has been characterized only in very vague terms. We show that the fractal dimension results for Hao's visualization frames directly translate to Jeffrey's CGR scheme.

  16. Nanopore-based fourth-generation DNA sequencing technology.

    Science.gov (United States)

    Feng, Yanxiao; Zhang, Yuechuan; Ying, Cuifeng; Wang, Deqiang; Du, Chunlei

    2015-02-01

    Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  17. Nanopore-based Fourth-generation DNA Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Yanxiao Feng

    2015-02-01

    Full Text Available Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

  18. Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing.

    Science.gov (United States)

    Tourlousse, Dieter M; Yoshiike, Satowa; Ohashi, Akiko; Matsukura, Satoko; Noda, Naohiro; Sekiguchi, Yuji

    2017-02-28

    High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. VoSeq: a voucher and DNA sequence web application.

    Directory of Open Access Journals (Sweden)

    Carlos Peña

    Full Text Available There is an ever growing number of molecular phylogenetic studies published, due to, in part, the advent of new techniques that allow cheap and quick DNA sequencing. Hence, the demand for relational databases with which to manage and annotate the amassing DNA sequences, genes, voucher specimens and associated biological data is increasing. In addition, a user-friendly interface is necessary for easy integration and management of the data stored in the database back-end. Available databases allow management of a wide variety of biological data. However, most database systems are not specifically constructed with the aim of being an organizational tool for researchers working in phylogenetic inference. We here report a new software facilitating easy management of voucher and sequence data, consisting of a relational database as back-end for a graphic user interface accessed via a web browser. The application, VoSeq, includes tools for creating molecular datasets of DNA or amino acid sequences ready to be used in commonly used phylogenetic software such as RAxML, TNT, MrBayes and PAUP, as well as for creating tables ready for publishing. It also has inbuilt BLAST capabilities against all DNA sequences stored in VoSeq as well as sequences in NCBI GenBank. By using mash-ups and calls to web services, VoSeq allows easy integration with public services such as Yahoo! Maps, Flickr, Encyclopedia of Life (EOL and GBIF (by generating data-dumps that can be processed with GBIF's Integrated Publishing Toolkit.

  20. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    Energy Technology Data Exchange (ETDEWEB)

    Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  1. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    International Nuclear Information System (INIS)

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-01-01

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  2. Detection of inter-spread repeat sequence in genomic DNA sequence.

    Science.gov (United States)

    Murakami, Hiroo; Sugaya, Nobuyoshi; Sato, Makihiko; Imaizumi, Akira; Aburatani, Sachiyo; Horimoto, Katsuhisa

    2004-01-01

    Various types of periodic patterns in nucleotide sequences are known to be very abundant in a genomic DNA sequence, and to play important biological roles such as gene expression, genome structural stabilization, and recombination. We present a new method, named "STEPSTONE", to find a specific periodic pattern of repeat sequence, inter-spread repeat, in which the tandem repeats of the conserved and the not-conserved regions appear periodically. In our method, at first, the data on periods of short repeat sequences found in a target sequence are stored as a hash data, and then are selected by application of an auto-correlation test in time series analysis. Among the statistically selected sequences, the inter-spread repeats are obtained by usual alignment procedures through two steps. To test the performance of our method, we examined the inter-spread repeats in Mycobacterium tuberculosis and Zamia paucijuga genomic sequences. As a result, our method exactly detected the repeats in the two sequences, being useful for identifying systematically the inter-spread repeats in DNA sequence.

  3. High-Throughput DNA sequencing of ancient wood.

    Science.gov (United States)

    Wagner, Stefanie; Lagane, Frédéric; Seguin-Orlando, Andaine; Schubert, Mikkel; Leroy, Thibault; Guichoux, Erwan; Chancerel, Emilie; Bech-Hebelstrup, Inger; Bernard, Vincent; Billard, Cyrille; Billaud, Yves; Bolliger, Matthias; Croutsch, Christophe; Čufar, Katarina; Eynaud, Frédérique; Heussner, Karl Uwe; Köninger, Joachim; Langenegger, Fabien; Leroy, Frédéric; Lima, Christine; Martinelli, Nicoletta; Momber, Garry; Billamboz, André; Nelle, Oliver; Palomo, Antoni; Piqué, Raquel; Ramstein, Marianne; Schweichel, Roswitha; Stäuble, Harald; Tegel, Willy; Terradas, Xavier; Verdin, Florence; Plomion, Christophe; Kremer, Antoine; Orlando, Ludovic

    2018-03-01

    Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high-throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long-term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human-induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro-evolutionary response of trees to climate change and human forest management. © 2018 John Wiley & Sons Ltd.

  4. Biomass characteristics in three sequencing batch reactors treating a wastewater containing synthetic organic chemicals

    DEFF Research Database (Denmark)

    Hu, Z.Q.; Ferraina, R.A.; Ericson, J.F.

    2005-01-01

    The physical and biochemical characteristics of the biomass in three lab-scale sequencing batch reactors (SBR) treating a synthetic wastewater at a 20-day target solids retention time (SRT) were investigated. The synthetic wastewater feed contained biogenic compounds and 22 organic priming...... compounds, chosen to represent a wide variety of chemical structures with different N, P and S functional groups. At a two-day hydraulic retention time (HRT), the oxidation-reduction potential (ORP) cycled between -100 (anoxic) and 100mV (aerobic) in the anoxic/aerobic SBR, while it remained in a range...... of 126 +/- 18 and 249 +/- 18 mV in the aerobic sequencing batch biofilm reactor (SBBR) and the aerobic SBR reactor, respectively. A granular activated sludge with excellent settleability (SVI = 98 +/- 31 L mg(-1)) developed only in the anoxic/aerobic SBR, compared to a bulky sludge with poor settling...

  5. Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

    Science.gov (United States)

    Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

    2018-01-10

    Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

  6. Micropatterning stretched and aligned DNA for sequence-specific nanolithography

    Science.gov (United States)

    Petit, Cecilia Anna Paulette

    Techniques for fabricating nanostructured materials can be categorized as either "top-down" or "bottom-up". Top-down techniques use lithography and contact printing to create patterned surfaces and microfluidic channels that can corral and organize nanoscale structures, such as molecules and nanorods in contrast; bottom-up techniques use self-assembly or molecular recognition to direct the organization of materials. A central goal in nanotechnology is the integration of bottom-up and top-down assembly strategies for materials development, device design; and process integration. With this goal in mind, we have developed strategies that will allow this integration by using DNA as a template for nanofabrication; two top-down approaches allow the placement of these templates, while the bottom-up technique uses the specific sequence of bases to pattern materials along each strand of DNA. Our first top-down approach, termed combing of molecules in microchannels (COMMIC), produces microscopic patterns of stretched and aligned molecules of DNA on surfaces. This process consists of passing an air-water interface over end adsorbed molecules inside microfabricated channels. The geometry of the microchannel directs the placement of the DNA molecules, while the geometry of the airwater interface directs the local orientation and curvature of the molecules. We developed another top-down strategy for creating micropatterns of stretched and aligned DNA using surface chemistry. Because DNA stretching occurs on hydrophobic surfaces, this technique uses photolithography to pattern vinyl-terminated silanes on glass When these surface-, are immersed in DNA solution, molecules adhere preferentially to the silanized areas. This approach has also proven useful in patterning protein for cell adhesion studies. Finally, we describe the use of these stretched and aligned molecules of DNA as templates for the subsequent bottom-up construction of hetero-structures through hybridization

  7. The influence of DNA sequence on epigenome-induced pathologies

    Directory of Open Access Journals (Sweden)

    Meagher Richard B

    2012-07-01

    Full Text Available Abstract Clear cause-and-effect relationships are commonly established between genotype and the inherited risk of acquiring human and plant diseases and aberrant phenotypes. By contrast, few such cause-and-effect relationships are established linking a chromatin structure (that is, the epitype with the transgenerational risk of acquiring a disease or abnormal phenotype. It is not entirely clear how epitypes are inherited from parent to offspring as populations evolve, even though epigenetics is proposed to be fundamental to evolution and the likelihood of acquiring many diseases. This article explores the hypothesis that, for transgenerationally inherited chromatin structures, “genotype predisposes epitype”, and that epitype functions as a modifier of gene expression within the classical central dogma of molecular biology. Evidence for the causal contribution of genotype to inherited epitypes and epigenetic risk comes primarily from two different kinds of studies discussed herein. The first and direct method of research proceeds by the examination of the transgenerational inheritance of epitype and the penetrance of phenotype among genetically related individuals. The second approach identifies epitypes that are duplicated (as DNA sequences are duplicated and evolutionarily conserved among repeated patterns in the DNA sequence. The body of this article summarizes particularly robust examples of these studies from humans, mice, Arabidopsis, and other organisms. The bulk of the data from both areas of research support the hypothesis that genotypes predispose the likelihood of displaying various epitypes, but for only a few classes of epitype. This analysis suggests that renewed efforts are needed in identifying polymorphic DNA sequences that determine variable nucleosome positioning and DNA methylation as the primary cause of inherited epigenome-induced pathologies. By contrast, there is very little evidence that DNA sequence directly

  8. Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera).

    Science.gov (United States)

    Barragán, M J L; Martínez, S; Marchal, J A; Fernández, R; Bullejos, M; Díaz de la Guardia, R; Sánchez, A

    2003-09-01

    This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.

  9. Early Lyme disease with spirochetemia - diagnosed by DNA sequencing

    Directory of Open Access Journals (Sweden)

    Jones William

    2010-11-01

    Full Text Available Abstract Background A sensitive and analytically specific nucleic acid amplification test (NAAT is valuable in confirming the diagnosis of early Lyme disease at the stage of spirochetemia. Findings Venous blood drawn from patients with clinical presentations of Lyme disease was tested for the standard 2-tier screen and Western Blot serology assay for Lyme disease, and also by a nested polymerase chain reaction (PCR for B. burgdorferi sensu lato 16S ribosomal DNA. The PCR amplicon was sequenced for B. burgdorferi genomic DNA validation. A total of 130 patients visiting emergency room (ER or Walk-in clinic (WALKIN, and 333 patients referred through the private physicians' offices were studied. While 5.4% of the ER/WALKIN patients showed DNA evidence of spirochetemia, none (0% of the patients referred from private physicians' offices were DNA-positive. In contrast, while 8.4% of the patients referred from private physicians' offices were positive for the 2-tier Lyme serology assay, only 1.5% of the ER/WALKIN patients were positive for this antibody test. The 2-tier serology assay missed 85.7% of the cases of early Lyme disease with spirochetemia. The latter diagnosis was confirmed by DNA sequencing. Conclusion Nested PCR followed by automated DNA sequencing is a valuable supplement to the standard 2-tier antibody assay in the diagnosis of early Lyme disease with spirochetemia. The best time to test for Lyme spirochetemia is when the patients living in the Lyme disease endemic areas develop unexplained symptoms or clinical manifestations that are consistent with Lyme disease early in the course of their illness.

  10. Prediction of fine-tuned promoter activity from DNA sequence.

    Science.gov (United States)

    Siwo, Geoffrey; Rider, Andrew; Tan, Asako; Pinapati, Richard; Emrich, Scott; Chawla, Nitesh; Ferdig, Michael

    2016-01-01

    The quantitative prediction of transcriptional activity of genes using promoter sequence is fundamental to the engineering of biological systems for industrial purposes and understanding the natural variation in gene expression. To catalyze the development of new algorithms for this purpose, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) organized a community challenge seeking predictive models of promoter activity given normalized promoter activity data for 90 ribosomal protein promoters driving expression of a fluorescent reporter gene. By developing an unbiased modeling approach that performs an iterative search for predictive DNA sequence features using the frequencies of various k-mers, inferred DNA mechanical properties and spatial positions of promoter sequences, we achieved the best performer status in this challenge. The specific predictive features used in the model included the frequency of the nucleotide G, the length of polymeric tracts of T and TA, the frequencies of 6 distinct trinucleotides and 12 tetranucleotides, and the predicted protein deformability of the DNA sequence. Our method accurately predicted the activity of 20 natural variants of ribosomal protein promoters (Spearman correlation r = 0.73) as compared to 33 laboratory-mutated variants of the promoters (r = 0.57) in a test set that was hidden from participants. Notably, our model differed substantially from the rest in 2 main ways: i) it did not explicitly utilize transcription factor binding information implying that subtle DNA sequence features are highly associated with gene expression, and ii) it was entirely based on features extracted exclusively from the 100 bp region upstream from the translational start site demonstrating that this region encodes much of the overall promoter activity. The findings from this study have important implications for the engineering of predictable gene expression systems and the evolution of gene expression in naturally occurring

  11. Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

    Science.gov (United States)

    Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

    2010-01-01

    Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

  12. The DNA sequence, annotation and analysis of human chromosome 3

    DEFF Research Database (Denmark)

    Muzny, D.M.; Bolund, Lars; As part of the Chinese Human Genome Sequencing Consortium, E.T.A.L.

    2006-01-01

    chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well...... as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion...

  13. Human fetal globin DNA sequences suggest novel conversion event.

    OpenAIRE

    Stoeckert, C J; Collins, F S; Weissman, S M

    1984-01-01

    DNA sequencing studies of two recently cloned human A gamma globin alleles has revealed a number of base differences which are clustered in the large intron (IVS-2). One allele has a previously undescribed IVS-2 sequence. Most of the allelic differences can be explained as resulting from a gene conversion event involving G gamma as a donor. A novel feature of this event is that three G gamma-like regions occur interspersed among unconverted areas of the A gamma gene. We propose that an altern...

  14. Electron microscopic and physico-chemical studies of DNA complexes with synthetic oligopeptides: binding specificity and DNA compact structures.

    Science.gov (United States)

    Vengerov, Y Y; Semenov, T E; Surovaya, A N; Sidorova NYu; Streltsov, S A; Khorlin, A A; Zhuze, A L; Gursky, G V

    1988-10-01

    Binding to DNA of two synthetic peptides, Val-Thr-Thr-Val-Val-NH-NH-Dns and Thr-Val-Thr-Lys-Val-Gly-Thr-Lsy-Val-Gly-Thr-Val-Val-NH-NH-Dns (where Dns is a residue of 5-dimethylaminonaphthalene-1-sulfonic acid), has been studied by circular dichroism, electron microscopy and fluorescence methods. It has been found that these two peptides can self-associate in aqueous solution as follows from the fact that concentration-dependent changes are observed in the UV absorbance and fluorescence spectra. The two peptides can bind to DNA both in self-associated and monomeric forms. The pentapeptide in the beta-associated form binds more strongly to poly(dG).poly(dC) than to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) whereas the tridecapeptide exhibits an opposite order of preferences binding more strongly to poly[d(A-C)].poly[d(G-T)] and poly(dA).poly(dT) than to poly(dG).poly(dC). Binding is a cooperative process which is accompanied by the DNA compaction at peptide/DNA base pair ratios greater than 1. At the initial stage of the compaction process, the coalescence of DNA segments covered by bound peptide molecules leads to the formation of DNA loops stabilized by the interaction between peptide molecules bound to different DNA segments. Further increase in the peptide/DNA ratio leads to the formation of rod-like structures each consisting of two or more double-stranded DNA segments. The final stage of the compaction process involves folding of fibrillar macromolecular complexes into a globular structure containing only one DNA molecule.

  15. Effect of dephasing on DNA sequencing via transverse electronic transport

    Energy Technology Data Exchange (ETDEWEB)

    Zwolak, Michael [Los Alamos National Laboratory; Krems, Matt [NON LANL; Pershin, Yuriy V [NON LANL; Di Ventra, Massimiliano [NON LANL

    2009-01-01

    We study theoretically the effects of dephasing on DNA sequencing in a nanopore via transverse electronic transport. To do this, we couple classical molecular dynamics simulations with transport calculations using scattering theory. Previous studies, which did not include dephasing, have shown that by measuring the transverse current of a particular base multiple times, one can get distributions of currents for each base that are distinguishable. We introduce a dephasing parameter into transport calculations to simulate the effects of the ions and other fluctuations. These effects lower the overall magnitude of the current, but have little effect on the current distributions themselves. The results of this work further implicate that distinguishing DNA bases via transverse electronic transport has potential as a sequencing tool.

  16. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure tha...

  17. Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

    OpenAIRE

    Brumley, R L; Smith, L M

    1991-01-01

    A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The appl...

  18. Development of a defined-sequence DNA system for use in DNA misrepair studies

    International Nuclear Information System (INIS)

    Sutton, S.; Tobias, C.A.

    1984-01-01

    The authors have developed a system that allows them to study cellular DNA repair processes at the molecular level. In particular, the authors are using this system to examine the consequences of a misrepair of radiation-induced DNA damage, as a function of dose. The cells being used are specially engineered haploid yeast cells. Maintained in the cells, at one copy per cell, is a cen plasmid, a plasmid that behaves like a functional chromosome. This plasmid carries a small defined sequence of DNA from the E. coli lac z gene. It is this lac z region (called the alpha region) that serves as the target for radiation damage. Two copies of the complimentary portion of the lac z gene are integrated into the yeast genome. Irradiated cells are screened for possible mutation in the alpha region by testing the cells' ability to hydrolyze xgal, a lactose substrate. The DNA of interest is then extracted from the cells, sequenced, and the sequence is compared to that of the control. Unlike the usual defined-sequence DNA systems, theirs is an in vivo system. A disadvantage is the relatively high background mutation rate. Results achieved with this system, as well as future applications, are discussed

  19. ThreaDNA: predicting DNA mechanics' contribution to sequence selectivity of proteins along whole genomes.

    Science.gov (United States)

    Cevost, Jasmin; Vaillant, Cédric; Meyer, Sam; Rost, Burkhard

    2018-02-15

    Many DNA-binding proteins recognize their target sequences indirectly, by sensing DNA's response to mechanical distortion. ThreaDNA estimates this response based on high-resolution structures of the protein-DNA complex of interest. Implementing an efficient nanoscale modeling of DNA deformations involving essentially no adjustable parameters, it returns the profile of deformation energy along whole genomes, at base-pair resolution, within minutes on usual laptop/desktop computers. Our predictions can also be easily combined with estimations of direct selectivity through a generalized form of position-weight-matrices. The formalism of ThreaDNA is accessible to a wide audience. We demonstrate the importance of indirect readout for the nucleosome as well as the bacterial regulators Fis and CRP. Combined with the direct contribution provided by usual sequence motifs, it significantly improves the prediction of sequence selectivity, and allows quantifying the two distinct physical mechanisms underlying it. Python software available at bioinfo.insa-lyon.fr, natively executable on Linux/MacOS systems with a user-friendly graphical interface. Galaxy webserver version available. sam.meyer@insa-lyon.fr. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  20. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  1. Inferring relative proportions of DNA variants from sequencing electropherograms.

    Science.gov (United States)

    Carr, I M; Robinson, J I; Dimitriou, R; Markham, A F; Morgan, A W; Bonthron, D T

    2009-12-15

    Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. msjimc@leeds.ac.uk Supplementary data are available at Bioinformatics online.

  2. Targeted DNA methylation analysis by next-generation sequencing.

    Science.gov (United States)

    Masser, Dustin R; Stanford, David R; Freeman, Willard M

    2015-02-24

    The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

  3. A unique DNA repair and recombination gene (recN) sequence for ...

    Indian Academy of Sciences (India)

    2013-04-23

    Apr 23, 2013 ... A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences. AUNDY KUMAR. 1,*, THEKKAN PUTHIYAVEEDU PRAMEELA.

  4. SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology

    DEFF Research Database (Denmark)

    Kim, Se Hyeuk; Cavaleiro, Mafalda; Rennig, Maja

    2016-01-01

    DNA vectors serve to maintain and select recombinant DNA in cell factories, and as design complexity increases, there is a greater need for well-characterized parts and methods for their assembly. Standards in synthetic biology are top priority, but standardizing molecular cloning contrasts...... flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard “SEVA linkers” for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson...

  5. Identification of Staphylococcal Enterotoxin B Sequences Important for Induction of Lymphocyte Proliferation by Using Synthetic Peptide Fragments of the Toxin

    Science.gov (United States)

    1994-08-01

    Staphylococcal Enterotoxin B Sequences STO=T Important for induction of lymphocyte proliferatio WPP8, WPPM,by using synthetic peptide fragments of the...Lymphocyte Proliferation by Using Synthetic Peptide Fragments of the Toxin MARTI JETT.I* ROGER NEILL,’ CHRISTOPHER WELCH,’ THOMAS BOYLE,’ EDWARD BERNTON...fragment of SEC (41) and for an amino- ment of endotoxic shock (42), induction of immunosuppres- terminal synthetic peptide of SEA (36). Another study

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  7. Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

    Science.gov (United States)

    Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

    2018-02-10

    High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second

  8. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

    Science.gov (United States)

    André, P; Kim, A; Khrapko, K; Thilly, W G

    1997-08-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

  9. Nanobiosensor for Detection and Quantification of DNA Sequences in Degraded Mixed Meats

    Directory of Open Access Journals (Sweden)

    M. E. Ali

    2011-01-01

    Full Text Available A novel class of nanobiosensor was developed by integrating a 27-nucleotide AluI fragment of swine cytochrome b (cytb gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP. The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic and AluI digested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

  10. Sequencing of mitochondrial HV1 and HV2 DNA with length heteroplasmy

    DEFF Research Database (Denmark)

    Rasmussen, E. Michael; Eriksen, Birthe; Larsen, Hans Jakob

    2003-01-01

    This study presents a fast method for sequencing the poly C/G regions in HV1 and HV2 in the mitochondrial DNA (mtDNA)......This study presents a fast method for sequencing the poly C/G regions in HV1 and HV2 in the mitochondrial DNA (mtDNA)...

  11. Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

    Science.gov (United States)

    M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

    2006-01-01

    Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

  12. Amplification of bleomycin-induced DNA cleavage at cytosine residues 3' to GGG sequences by pyrrole triamide.

    Science.gov (United States)

    Hiraku, Y; Oikawa, S; Kuroki, K; Sugiyama, H; Saito, I; Kawanishi, S

    2001-02-01

    We investigated the amplification of bleomycin-induced DNA cleavage by synthetic triamides containing N-methylpyrrole (Py) and/or N-methylimidazole (Im), PyPyPy, PyPyIm, PyImPy, and PyImIm, using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 and p53 genes. Peplomycin, a bleomycin analog, plus Fe(II) caused DNA cleavage at the 5'-GC-3' and 5'-GT-3' sequences (damaged bases are underlined). The addition of PyPyPy dramatically enhanced the cleavage, particularly at cytosine residues 3' to consecutive guanines. Alteration in the site specificity was not observed with other triamides (PyPyIm, PyImPy, and PyImIm). DNase I footprinting revealed that PyPyPy bound to the sites adjacent to the sites where DNA cleavage was enhanced by PyPyPy, and that PyPyPy enhanced DNase I-induced cleavage in GC-rich regions. These findings suggest that binding of PyPyPy to the DNA minor groove changes the DNA conformation to allow peplomycin to cleave DNA more efficiently at GC-rich sequences, resulting in intensive site-specific DNA cleavage particularly at cytosines at the 3'-side of polyguanines. The present study on amplifiers of antitumor drugs would appear to offer a novel approach to the establishment of more effective chemotherapy.

  13. Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.

    Science.gov (United States)

    Collins, Louise; Kaszuba, Michael; Fabre, John W

    2004-04-07

    The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.

  14. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    Science.gov (United States)

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-08-17

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells.

  15. PISMA: A Visual Representation of Motif Distribution in DNA Sequences

    Directory of Open Access Journals (Sweden)

    Rogelio Alcántara-Silva

    2017-03-01

    Full Text Available Background: Because the graphical presentation and analysis of motif distribution can provide insights for experimental hypothesis, PISMA aims at identifying motifs on DNA sequences, counting and showing them graphically. The motif length ranges from 2 to 10 bases, and the DNA sequences range up to 10 kb. The motif distribution is shown as a bar-code–like, as a gene-map–like, and as a transcript scheme. Results: We obtained graphical schemes of the CpG site distribution from 91 human papillomavirus genomes. Also, we present 2 analyses: one of DNA motifs associated with either methylation-resistant or methylation-sensitive CpG islands and another analysis of motifs associated with exosome RNA secretion. Availability and Implementation: PISMA is developed in Java; it is executable in any type of hardware and in diverse operating systems. PISMA is freely available to noncommercial users. The English version and the User Manual are provided in Supplementary Files 1 and 2, and a Spanish version is available at www.biomedicas.unam.mx/wp-content/software/pisma.zip and www.biomedicas.unam.mx/wp-content/pdf/manual/pisma.pdf .

  16. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  17. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  18. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

    Science.gov (United States)

    Marosy, Beth A.; Craig, Brian D.; Hetrick, Kurt N.; Witmer, P. Dane; Ling, Hua; Griffith, Sean M.; Myers, Ben; Ostrander, Elaine A.; Stanford, Janet L.; Brody, Lawrence C.; Doheny, Kimberly F.

    2016-01-01

    This unit describes a protocol for generating exome enriched sequencing libraries using DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) samples. Utilizing commercially available kits, we present a low input FFPE workflow starting with 50ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution targeted selection for exons, followed by sequencing using the Illumina next generation short read sequencing platform. PMID:28075488

  19. DNA Sequencing as a Tool to Monitor Marine Ecological Status

    Directory of Open Access Journals (Sweden)

    Kelly D. Goodwin

    2017-05-01

    Full Text Available Many ocean policies mandate integrated, ecosystem-based approaches to marine monitoring, driving a global need for efficient, low-cost bioindicators of marine ecological quality. Most traditional methods to assess biological quality rely on specialized expertise to provide visual identification of a limited set of specific taxonomic groups, a time-consuming process that can provide a narrow view of ecological status. In addition, microbial assemblages drive food webs but are not amenable to visual inspection and thus are largely excluded from detailed inventory. Molecular-based assessments of biodiversity and ecosystem function offer advantages over traditional methods and are increasingly being generated for a suite of taxa using a “microbes to mammals” or “barcodes to biomes” approach. Progress in these efforts coupled with continued improvements in high-throughput sequencing and bioinformatics pave the way for sequence data to be employed in formal integrated ecosystem evaluation, including food web assessments, as called for in the European Union Marine Strategy Framework Directive. DNA sequencing of bioindicators, both traditional (e.g., benthic macroinvertebrates, ichthyoplankton and emerging (e.g., microbial assemblages, fish via eDNA, promises to improve assessment of marine biological quality by increasing the breadth, depth, and throughput of information and by reducing costs and reliance on specialized taxonomic expertise.

  20. A MapReduce Framework for DNA Sequencing Data Processing

    Directory of Open Access Journals (Sweden)

    Samy Ghoneimy

    2016-12-01

    Full Text Available Genomics and Next Generation Sequencers (NGS like Illumina Hiseq produce data in the order of ‎‎200 billion base pairs in a single one-week run for a 60x human genome coverage, which ‎requires modern high-throughput experimental technologies that can ‎only be tackled with high performance computing (HPC and specialized software algorithms called ‎‎“short read aligners”. This paper focuses on the implementation of the DNA sequencing as a set of MapReduce programs that will accept a DNA data set as a FASTQ file and finally generate a VCF (variant call format file, which has variants for a given DNA data set. In this paper MapReduce/Hadoop along with Burrows-Wheeler Aligner (BWA, Sequence Alignment/Map (SAM ‎tools, are fully utilized to provide various utilities for manipulating alignments, including sorting, merging, indexing, ‎and generating alignments. The Map-Sort-Reduce process is designed to be suited for a Hadoop framework in ‎which each cluster is a traditional N-node Hadoop cluster to utilize all of the Hadoop features like HDFS, program ‎management and fault tolerance. The Map step performs multiple instances of the short read alignment algorithm ‎‎(BoWTie that run in parallel in Hadoop. The ordered list of the sequence reads are used as input tuples and the ‎output tuples are the alignments of the short reads. In the Reduce step many parallel instances of the Short ‎Oligonucleotide Analysis Package for SNP (SOAPsnp algorithm run in the cluster. Input tuples are sorted ‎alignments for a partition and the output tuples are SNP calls. Results are stored via HDFS, and then archived in ‎SOAPsnp format. ‎ The proposed framework enables extremely fast discovering somatic mutations, inferring population genetical ‎parameters, and performing association tests directly based on sequencing data without explicit genotyping or ‎linkage-based imputation. It also demonstrate that this method achieves comparable

  1. Peptide Synthesis on a Next-Generation DNA Sequencing Platform.

    Science.gov (United States)

    Svensen, Nina; Peersen, Olve B; Jaffrey, Samie R

    2016-09-02

    Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) . The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein- or small molecule-binding aptamers. We used the mRNA-display approach to synthesize flow-cell-tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide-based assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Retroviral DNA Sequences as a Means for Determining Ancient Diets.

    Directory of Open Access Journals (Sweden)

    Jessica I Rivera-Perez

    Full Text Available For ages, specialists from varying fields have studied the diets of the primeval inhabitants of our planet, detecting diet remains in archaeological specimens using a range of morphological and biochemical methods. As of recent, metagenomic ancient DNA studies have allowed for the comparison of the fecal and gut microbiomes associated to archaeological specimens from various regions of the world; however the complex dynamics represented in those microbial communities still remain unclear. Theoretically, similar to eukaryote DNA the presence of genes from key microbes or enzymes, as well as the presence of DNA from viruses specific to key organisms, may suggest the ingestion of specific diet components. In this study we demonstrate that ancient virus DNA obtained from coprolites also provides information reconstructing the host's diet, as inferred from sequences obtained from pre-Columbian coprolites. This depicts a novel and reliable approach to determine new components as well as validate the previously suggested diets of extinct cultures and animals. Furthermore, to our knowledge this represents the first description of the eukaryotic viral diversity found in paleofaeces belonging to pre-Columbian cultures.

  3. Mitochondrial DNA sequencing of cat hair: an informative forensic tool.

    Science.gov (United States)

    Tarditi, Christy R; Grahn, Robert A; Evans, Jeffrey J; Kurushima, Jennifer D; Lyons, Leslie A

    2011-01-01

    Approximately 81.7 million cats are in 37.5 million U.S. households. Shed fur can be criminal evidence because of transfer to victims, suspects, and/or their belongings. To improve cat hairs as forensic evidence, the mtDNA control region from single hairs, with and without root tags, was sequenced. A dataset of a 402-bp control region segment from 174 random-bred cats representing four U.S. geographic areas was generated to determine the informativeness of the mtDNA region. Thirty-two mtDNA mitotypes were observed ranging in frequencies from 0.6-27%. Four common types occurred in all populations. Low heteroplasmy, 1.7%, was determined. Unique mitotypes were found in 18 individuals, 10.3% of the population studied. The calculated discrimination power implied that 8.3 of 10 randomly selected individuals can be excluded by this region. The genetic characteristics of the region and the generated dataset support the use of this cat mtDNA region in forensic applications. 2010 American Academy of Forensic Sciences. Published 2010. This article is a U.S. Government work and is in the public domain in the U.S.A.

  4. Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing.

    Science.gov (United States)

    Vaca-Paniagua, Felipe; Oliver, Javier; Nogueira da Costa, Andre; Merle, Philippe; McKay, James; Herceg, Zdenko; Holmila, Reetta

    2015-01-01

    To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

  5. A Simulation of DNA Sequencing Utilizing 3M Post-It[R] Notes

    Science.gov (United States)

    Christensen, Doug

    2009-01-01

    An inexpensive and equipment free approach to teaching the technical aspects of DNA sequencing. The activity described requires an instructor with a familiarity of DNA sequencing technology but provides a straight forward method of teaching the technical aspects of sequencing in the absence of expensive sequencing equipment. The final sequence…

  6. Entropy and long-range correlations in DNA sequences.

    Science.gov (United States)

    Melnik, S S; Usatenko, O V

    2014-12-01

    We analyze the structure of DNA molecules of different organisms by using the additive Markov chain approach. Transforming nucleotide sequences into binary strings, we perform statistical analysis of the corresponding "texts". We develop the theory of N-step additive binary stationary ergodic Markov chains and analyze their differential entropy. Supposing that the correlations are weak we express the conditional probability function of the chain by means of the pair correlation function and represent the entropy as a functional of the pair correlator. Since the model uses two point correlators instead of probability of block occurring, it makes possible to calculate the entropy of subsequences at much longer distances than with the use of the standard methods. We utilize the obtained analytical result for numerical evaluation of the entropy of coarse-grained DNA texts. We believe that the entropy study can be used for biological classification of living species. Copyright © 2014. Published by Elsevier Ltd.

  7. Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII.

    Science.gov (United States)

    Vossen, Rolf H A M; Buermans, Henk P J

    2017-01-01

    Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.

  8. New scoring schema for finding motifs in DNA Sequences

    Directory of Open Access Journals (Sweden)

    Nowzari-Dalini Abbas

    2009-03-01

    Full Text Available Abstract Background Pattern discovery in DNA sequences is one of the most fundamental problems in molecular biology with important applications in finding regulatory signals and transcription factor binding sites. An important task in this problem is to search (or predict known binding sites in a new DNA sequence. For this reason, all subsequences of the given DNA sequence are scored based on an scoring function and the prediction is done by selecting the best score. By assuming no dependency between binding site base positions, most of the available tools for known binding site prediction are designed. Recently Tomovic and Oakeley investigated the statistical basis for either a claim of dependence or independence, to determine whether such a claim is generally true, and they presented a scoring function for binding site prediction based on the dependency between binding site base positions. Our primary objective is to investigate the scoring functions which can be used in known binding site prediction based on the assumption of dependency or independency in binding site base positions. Results We propose a new scoring function based on the dependency between all positions in biding site base positions. This scoring function uses joint information content and mutual information as a measure of dependency between positions in transcription factor binding site. Our method for modeling dependencies is simply an extension of position independency methods. We evaluate our new scoring function on the real data sets extracted from JASPAR and TRANSFAC data bases, and compare the obtained results with two other well known scoring functions. Conclusion The results demonstrate that the new approach improves known binding site discovery and show that the joint information content and mutual information provide a better and more general criterion to investigate the relationships between positions in the TFBS. Our scoring function is formulated by simple

  9. Synthetic

    Directory of Open Access Journals (Sweden)

    Anna Maria Manferdini

    2010-06-01

    Full Text Available Traditionally materials have been associated with a series of physical properties that can be used as inputs to production and manufacturing. Recently we witnessed an interest in materials considered not only as ‘true matter’, but also as new breeds where geometry, texture, tooling and finish are able to provoke new sensations when they are applied to a substance. These artificial materials can be described as synthetic because they are the outcome of various qualities that are not necessarily true to the original matter, but they are the combination of two or more parts, whether by design or by natural processes. The aim of this paper is to investigate the potential of architectural surfaces to produce effects through the invention of new breeds of artificial matter, using micro-scale details derived from Nature as an inspiration.

  10. DNA interaction with platinum-based cytostatics revealed by DNA sequencing.

    Science.gov (United States)

    Smerkova, Kristyna; Vaculovic, Tomas; Vaculovicova, Marketa; Kynicky, Jindrich; Brtnicky, Martin; Eckschlager, Tomas; Stiborova, Marie; Hubalek, Jaromir; Adam, Vojtech

    2017-12-15

    The main mechanism of action of platinum-based cytostatic drugs - cisplatin, oxaliplatin and carboplatin - is the formation of DNA cross-links, which restricts the transcription due to the disability of DNA to enter the active site of the polymerase. The polymerase chain reaction (PCR) was employed as a simplified model of the amplification process in the cell nucleus. PCR with fluorescently labelled dideoxynucleotides commonly employed for DNA sequencing was used to monitor the effect of platinum-based cytostatics on DNA in terms of decrease in labeling efficiency dependent on a presence of the DNA-drug cross-link. It was found that significantly different amounts of the drugs - cisplatin (0.21 μg/mL), oxaliplatin (5.23 μg/mL), and carboplatin (71.11 μg/mL) - were required to cause the same quenching effect (50%) on the fluorescent labelling of 50 μg/mL of DNA. Moreover, it was found that even though the amounts of the drugs was applied to the reaction mixture differing by several orders of magnitude, the amount of incorporated platinum, quantified by inductively coupled plasma mass spectrometry, was in all cases at the level of tenths of μg per 5 μg of DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 18S rDNA sequences and the holometabolous insects.

    Science.gov (United States)

    Carmean, D; Kimsey, L S; Berbee, M L

    1992-12-01

    The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    Science.gov (United States)

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  13. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    Science.gov (United States)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. © 2016 Elsevier Inc. All rights reserved.

  14. Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots.

    Science.gov (United States)

    Kawano, Ryuji

    2018-02-19

    A molecular robot is a next-generation biochemical machine that imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on recent research on synthetic ion channels and DNA computing technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which are embedded in artificial cell membranes (lipid bilayers), sense ambient ions or chemicals and import them. These artificial sensors are useful components for molecular robots with bodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot to function as gates. After the signal molecules arrive inside the molecular robot, they can operate DNA logic gates, which perform computations. These functions will be integrated into the intelligence and sensor sections of molecular robots. Soon, these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and in vivo diagnosis or therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Smart DNA Fabrication Using Sound Waves: Applying Acoustic Dispensing Technologies to Synthetic Biology.

    Science.gov (United States)

    Kanigowska, Paulina; Shen, Yue; Zheng, Yijing; Rosser, Susan; Cai, Yizhi

    2016-02-01

    Acoustic droplet ejection (ADE) technology uses focused acoustic energy to transfer nanoliter-scale liquid droplets with high precision and accuracy. This noncontact, tipless, low-volume dispensing technology minimizes the possibility of cross-contamination and potentially reduces the costs of reagents and consumables. To date, acoustic dispensers have mainly been used in screening libraries of compounds. In this paper, we describe the first application of this powerful technology to the rapidly developing field of synthetic biology, for DNA synthesis and assembly at the nanoliter scale using a Labcyte Echo 550 acoustic dispenser. We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold. We envision that acoustic dispensing will become an instrumental technology in synthetic biology, in particular in the era of DNA foundries. © 2015 Society for Laboratory Automation and Screening.

  16. Recent progress in atomistic simulation of electrical current DNA sequencing.

    Science.gov (United States)

    Kim, Han Seul; Kim, Yong-Hoon

    2015-07-15

    We review recent advances in the DNA sequencing method based on measurements of transverse electrical currents. Device configurations proposed in the literature are classified according to whether the molecular fingerprints appear as the major (Mode I) or perturbing (Mode II) current signals. Scanning tunneling microscope and tunneling electrode gap configurations belong to the former category, while the nanochannels with or without an embedded nanopore belong to the latter. The molecular sensing mechanisms of Modes I and II roughly correspond to the electron tunneling and electrochemical gating, respectively. Special emphasis will be given on the computer simulation studies, which have been playing a critical role in the initiation and development of the field. We also highlight low-dimensional nanomaterials such as carbon nanotubes, graphene, and graphene nanoribbons that allow the novel Mode II approach. Finally, several issues in previous computational studies are discussed, which points to future research directions toward more reliable simulation of electrical current DNA sequencing devices. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Targeted bisulfite sequencing of the dynamic DNA methylome.

    Science.gov (United States)

    Ziller, Michael J; Stamenova, Elena K; Gu, Hongcang; Gnirke, Andreas; Meissner, Alexander

    2016-01-01

    The ability to measure DNA methylation precisely and efficiently continues to drive our understanding of this modification in development and disease. Whole genome bisulfite sequencing has the advantage of theoretically capturing all cytosines in the genome at single-nucleotide resolution, but it has a number of significant practical drawbacks that become amplified with increasing sample numbers. All other technologies capture only a fraction of the cytosines that show dynamic regulation across cell and tissue types. Here, we present a novel hybrid selection design focusing on loci with dynamic methylation that captures a large number of differentially methylated gene-regulatory elements. We benchmarked this assay against matched whole genome data and profiled 25 human tissue samples to explore its ability to detect differentially methylated regions. Our target capture design fills a major gap left by all other assays that exist to map DNA methylation. It maintains the ability to link cytosine methylation to genetic differences, the single-base resolution and the analysis of neighboring cytosines while notably reducing the cost per sample by focusing the sequencing effort on the most informative and relevant regions of the genome.

  18. Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus.

    Science.gov (United States)

    Hanotte, O; Burke, T; Armour, J A; Jeffreys, A J

    1991-04-01

    We report here for the first time the large-scale isolation of hypervariable minisatellite DNA sequences from a non-human species, the Indian peafowl (Pavo cristatus). A size-selected genomic DNA fraction, rich in hypervariable minisatellites, was cloned into Charomid 9-36. This library was screened using two multilocus hypervariable probes, 33.6 and 33.15 and also, in a "probe-walking" approach, with five of the peafowl minisatellites initially isolated. Forty-eight positively hybridizing clones were characterized and found to originate from 30 different loci, 18 of which were polymorphic. Five of these variable minisatellite loci were studied further. They all showed Mendelian inheritance. The heterozygosities of these loci were relatively low (range 22-78%) in comparison with those of previously cloned human loci, as expected in view of inbreeding in our semicaptive study population. No new length allele mutations were observed in families and the mean mutation rate per locus is low (less than 0.004, 95% confidence maximum). These loci were also investigated by cross-species hybridization in related taxa. The ability of the probes to detect hypervariable sequences in other species within the same avian family was found to vary, from those probes that are species-specific to those that are apparently general to the family. We also illustrate the potential usefulness of these probes for paternity analysis in a study of sexual selection, and discuss the general application of specific hypervariable probes in behavioral and evolutionary studies.

  19. Effect of intercalator substituent and nucleotide sequence on the stability of DNA- and RNA-naphthalimide complexes.

    Science.gov (United States)

    Johnson, Charles A; Hudson, Graham A; Hardebeck, Laura K E; Jolley, Elizabeth A; Ren, Yi; Lewis, Michael; Znosko, Brent M

    2015-07-01

    DNA intercalators are commonly used as anti-cancer and anti-tumor agents. As a result, it is imperative to understand how changes in intercalator structure affect binding affinity to DNA. Amonafide and mitonafide, two naphthalimide derivatives that are active against HeLa and KB cells in vitro, were previously shown to intercalate into DNA. Here, a systematic study was undertaken to change the 3-substituent on the aromatic intercalator 1,8-naphthalimide to determine how 11 different functional groups with a variety of physical and electronic properties affect binding of the naphthalimide to DNA and RNA duplexes of different sequence compositions and lengths. Wavelength scans, NMR titrations, and circular dichroism were used to investigate the binding mode of 1,8-naphthalimide derivatives to short synthetic DNA. Optical melting experiments were used to measure the change in melting temperature of the DNA and RNA duplexes due to intercalation, which ranged from 0 to 19.4°C. Thermal stabilities were affected by changing the substituent, and several patterns and idiosyncrasies were identified. By systematically varying the 3-substituent, the binding strength of the same derivative to various DNA and RNA duplexes was compared. The binding strength of different derivatives to the same DNA and RNA sequences was also compared. The results of these comparisons shed light on the complexities of site specificity and binding strength in DNA-intercalator complexes. For example, the consequences of adding a 5'-TpG-3' or 5'-GpT-3' step to a duplex is dependent on the sequence composition of the duplex. When added to a poly-AT duplex, naphthalimide binding was enhanced by 5.6-11.5°C, but when added to a poly-GC duplex, naphthalimide binding was diminished by 3.2-6.9°C. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. A novel method for comparative analysis of DNA sequences by Ramanujan-Fourier transform.

    Science.gov (United States)

    Yin, Changchuan; Yin, Xuemeng E; Wang, Jiasong

    2014-12-01

    Alignment-free sequence analysis approaches provide important alternatives over multiple sequence alignment (MSA) in biological sequence analysis because alignment-free approaches have low computation complexity and are not dependent on high level of sequence identity. However, most of the existing alignment-free methods do not employ true full information content of sequences and thus can not accurately reveal similarities and differences among DNA sequences. We present a novel alignment-free computational method for sequence analysis based on Ramanujan-Fourier transform (RFT), in which complete information of DNA sequences is retained. We represent DNA sequences as four binary indicator sequences and apply RFT on the indicator sequences to convert them into frequency domain. The Euclidean distance of the complete RFT coefficients of DNA sequences are used as similarity measures. To address the different lengths of RFT coefficients in Euclidean space, we pad zeros to short DNA binary sequences so that the binary sequences equal the longest length in the comparison sequence data. Thus, the DNA sequences are compared in the same dimensional frequency space without information loss. We demonstrate the usefulness of the proposed method by presenting experimental results on hierarchical clustering of genes and genomes. The proposed method opens a new channel to biological sequence analysis, classification, and structural module identification.

  1. Rapid infectious disease identification by next-generation DNA sequencing.

    Science.gov (United States)

    Ellis, Jeremy E; Missan, Dara S; Shabilla, Matthew; Martinez, Delyn; Fry, Stephen E

    2017-07-01

    Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties

    Directory of Open Access Journals (Sweden)

    Gaofeng Pan

    2018-02-01

    Full Text Available DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods—especially machine learning methods—have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k-gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria—area under the receiver operating characteristic curve (AUC, Matthew’s correlation coefficient (MCC, accuracy (ACC, sensitivity (SN, and specificity—are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

  3. A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties.

    Science.gov (United States)

    Pan, Gaofeng; Jiang, Limin; Tang, Jijun; Guo, Fei

    2018-02-08

    DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods-especially machine learning methods-have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k -gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria-area under the receiver operating characteristic curve (AUC), Matthew's correlation coefficient (MCC), accuracy (ACC), sensitivity (SN), and specificity-are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

  4. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus

  5. The evolution processes of DNA sequences, languages and carols

    Science.gov (United States)

    Hauck, Jürgen; Henkel, Dorothea; Mika, Klaus

    2001-04-01

    The sequences of bases A, T, C and G of about 100 enolase, secA and cytochrome DNA were analyzed for attractive or repulsive interactions by the numbers T 1,T 2,T 3; r of nearest, next-nearest and third neighbor bases of the same kind and the concentration r=other bases/analyzed base. The area of possible T1, T2 values is limited by the linear borders T 2=2T 1-2, T 2=0 or T1=0 for clustering, attractive or repulsive interactions and the border T2=-2 T1+2(2- r) for a variation from repulsive to attractive interactions at r⩽2. Clustering is preferred by most bases in sequences of enolases and secA’ s. Major deviations with repulsive interactions of some bases are observed for archaea bacteria in secA and for highly developed animals and the human species in enolase sequences. The borders of the structure map for enthalpy stabilized structures with maximum interactions are approached in few cases. Most letters of the natural languages and some music notes are at the borders of the structure map.

  6. FastGroup: A program to dereplicate libraries of 16S rDNA sequences

    Directory of Open Access Journals (Sweden)

    Rohwer Forest

    2001-10-01

    Full Text Available Abstract Background Ribosomal 16S DNA sequences are an essential tool for identifying and classifying microbes. High-throughput DNA sequencing now makes it economically possible to produce very large datasets of 16S rDNA sequences in short time periods, necessitating new computer tools for analyses. Here we describe FastGroup, a Java program designed to dereplicate libraries of 16S rDNA sequences. By dereplication we mean to: 1 compare all the sequences in a data set to each other, 2 group similar sequences together, and 3 output a representative sequence from each group. In this way, duplicate sequences are removed from a library. Results FastGroup was tested using a library of single-pass, bacterial 16S rDNA sequences cloned from coral-associated bacteria. We found that the optimal strategy for dereplicating these sequences was to: 1 trim ambiguous bases from the 5' end of the sequences and all sequence 3' of the conserved Bact517 site, 2 match the sequences from the 3' end, and 3 group sequences >=97% identical to each other. Conclusions The FastGroup program simplifies the dereplication of 16S rDNA sequence libraries and prepares the raw sequences for subsequent analyses.

  7. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters......, which brings independent evidence for the lateral gene transfer in the genome of T.maritima, The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms...

  8. RDNAnalyzer: A tool for DNA secondary structure prediction and sequence analysis.

    Science.gov (United States)

    Afzal, Muhammad; Shahid, Ahmad Ali; Shehzadi, Abida; Nadeem, Shahid; Husnain, Tayyab

    2012-01-01

    RDNAnalyzer is an innovative computer based tool designed for DNA secondary structure prediction and sequence analysis. It can randomly generate the DNA sequence or user can upload the sequences of their own interest in RAW format. It uses and extends the Nussinov dynamic programming algorithm and has various application for the sequence analysis. It predicts the DNA secondary structure and base pairings. It also provides the tools for routinely performed sequence analysis by the biological scientists such as DNA replication, reverse compliment generation, transcription, translation, sequence specific information as total number of nucleotide bases, ATGC base contents along with their respective percentages and sequence cleaner. RDNAnalyzer is a unique tool developed in Microsoft Visual Studio 2008 using Microsoft Visual C# and Windows Presentation Foundation and provides user friendly environment for sequence analysis. It is freely available. http://www.cemb.edu.pk/sw.html RDNAnalyzer - Random DNA Analyser, GUI - Graphical user interface, XAML - Extensible Application Markup Language.

  9. Partial DNA sequencing of Douglas-fir cDNAs used in RFLP mapping

    Science.gov (United States)

    K.D. Jermstad; D.L. Bassoni; C.S. Kinlaw; D.B. Neale

    1998-01-01

    DNA sequences from 87 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) cDNA RFLP probes were determined. Sequences were submitted to the GenBank dbEST database and searched for similarity against nucleotide and protein databases using the BLASTn and BLASTx programs. Twenty-one sequences (24%) were assigned putative functions; 18 of which...

  10. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human.

    Science.gov (United States)

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-02-16

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

  11. Synthetic peptides derived from the sequence of a lasso peptide microcin J25 show antibacterial activity.

    Science.gov (United States)

    Soudy, Rania; Wang, Liru; Kaur, Kamaljit

    2012-03-01

    Microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized antibacterial peptide with a unique lasso structure. The lasso structure, produced with the aid of two processing enzymes, provides exceptional stability to MccJ25. We report the synthesis of six peptides (1-6), derived from the MccJ25 sequence, that are designed to form folded conformation by disulfide bond formation and electrostatic or hydrophobic interactions. Two peptides (1 and 6) display good activity against Salmonella newport, and are the first synthetic derivatives of MccJ25 that are bactericidal. Peptide 1 displays potent activity against several Salmonella strains including two MccJ25 resistant strains. The solution conformation and the stability studies of the active peptides suggest that they do not fold into a lasso conformation and peptide 1 displays antimicrobial activity by inhibition of target cell respiration. Like MccJ25, the synthetic MccJ25 derivatives display minimal toxicity to mammalian cells suggesting that these peptides act specifically on bacterial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

    Science.gov (United States)

    Calce, Enrica; De Luca, Stefania

    2017-01-05

    Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

    Science.gov (United States)

    Sawaya, Sterling; Boocock, James; Black, Michael A; Gemmell, Neil J

    2015-01-28

    Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

  14. Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

    Science.gov (United States)

    Larkin, Joseph; Henley, Robert Y.; Jadhav, Vivek; Korlach, Jonas; Wanunu, Meni

    2017-12-01

    Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides—zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

  15. Programmed coherent coupling in a synthetic DNA-based excitonic circuit

    Science.gov (United States)

    Boulais, Étienne; Sawaya, Nicolas P. D.; Veneziano, Rémi; Andreoni, Alessio; Banal, James L.; Kondo, Toru; Mandal, Sarthak; Lin, Su; Schlau-Cohen, Gabriela S.; Woodbury, Neal W.; Yan, Hao; Aspuru-Guzik, Alán; Bathe, Mark

    2018-02-01

    Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer. We then use structure-based modelling and quantum dynamics to guide the rational design of higher-order synthetic circuits consisting of multiple discrete dye aggregates within a DX-tile. These programmed circuits exhibit excitonic transport properties with prominent circular dichroism, superradiance, and fast delocalized exciton transfer, consistent with our quantum dynamics predictions. This bottom-up strategy offers a versatile approach to the rational design of strongly coupled excitonic circuits using spatially organized dye aggregates for use in coherent nanoscale energy transport, artificial light-harvesting, and nanophotonics.

  16. SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology.

    Science.gov (United States)

    Kim, Se Hyeuk; Cavaleiro, Ana Mafalda; Rennig, Maja; Nørholm, Morten H H

    2016-10-21

    DNA vectors serve to maintain and select recombinant DNA in cell factories, and as design complexity increases, there is a greater need for well-characterized parts and methods for their assembly. Standards in synthetic biology are top priority, but standardizing molecular cloning contrasts flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard "SEVA linkers" for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson isothermal assembly, uracil excision cloning, and a nicking enzyme-based methodology we term SEVA cloning. SEVA cloning is a simplistic one-tube protocol for backbone swapping directly from plasmid stock solutions. We demonstrate the different performance of 30 plasmid backbones for small molecule and protein production and obtain more than 10-fold improvement from a four-gene biosynthetic pathway and 430-fold improvement with a difficult-to-express membrane protein. The standardized linkers and protocols add to the Standard European Vectors Architecture (SEVA) resource and are freely available to the synthetic biology community.

  17. Beyond DNA Sequencing in Space: Current and Future Omics Capabilities of the Biomolecule Sequencer Payload

    Science.gov (United States)

    Wallace, Sarah

    2017-01-01

    Why do we need a DNA sequencer to support the human exploration of space? (A) Operational environmental monitoring; (1) Identification of contaminating microbes, (2) Infectious disease diagnosis, (3) Reduce down mass (sample return for environmental monitoring, crew health, etc.). (B) Research; (1) Human, (2) Animal, (3) Microbes/Cell lines, (4) Plant. (C) Med Ops; (1) Response to countermeasures, (2) Radiation, (3) Real-time analysis can influence medical intervention. (C) Support astrobiology science investigations; (1) Technology superiorly suited to in situ nucleic acid-based life detection, (2) Functional testing for integration into robotics for extraplanetary exploration mission.

  18. Biomolecule Sequencer: Next-Generation DNA Sequencing Technology for In-Flight Environmental Monitoring, Research, and Beyond

    Science.gov (United States)

    Smith, David J.; Burton, Aaron; Castro-Wallace, Sarah; John, Kristen; Stahl, Sarah E.; Dworkin, Jason Peter; Lupisella, Mark L.

    2016-01-01

    On the International Space Station (ISS), technologies capable of rapid microbial identification and disease diagnostics are not currently available. NASA still relies upon sample return for comprehensive, molecular-based sample characterization. Next-generation DNA sequencing is a powerful approach for identifying microorganisms in air, water, and surfaces onboard spacecraft. The Biomolecule Sequencer payload, manifested to SpaceX-9 and scheduled on the Increment 4748 research plan (June 2016), will assess the functionality of a commercially-available next-generation DNA sequencer in the microgravity environment of ISS. The MinION device from Oxford Nanopore Technologies (Oxford, UK) measures picoamp changes in electrical current dependent on nucleotide sequences of the DNA strand migrating through nanopores in the system. The hardware is exceptionally small (9.5 x 3.2 x 1.6 cm), lightweight (120 grams), and powered only by a USB connection. For the ISS technology demonstration, the Biomolecule Sequencer will be powered by a Microsoft Surface Pro3. Ground-prepared samples containing lambda bacteriophage, Escherichia coli, and mouse genomic DNA, will be launched and stored frozen on the ISS until experiment initiation. Immediately prior to sequencing, a crew member will collect and thaw frozen DNA samples, connect the sequencer to the Surface Pro3, inject thawed samples into a MinION flow cell, and initiate sequencing. At the completion of the sequencing run, data will be downlinked for ground analysis. Identical, synchronous ground controls will be used for data comparisons to determine sequencer functionality, run-time sequence, current dynamics, and overall accuracy. We will present our latest results from the ISS flight experiment the first time DNA has ever been sequenced in space and discuss the many potential applications of the Biomolecule Sequencer for environmental monitoring, medical diagnostics, higher fidelity and more adaptable Space Biology Human

  19. An Efficient Approach to Mining Maximal Contiguous Frequent Patterns from Large DNA Sequence Databases

    Directory of Open Access Journals (Sweden)

    Md. Rezaul Karim

    2012-03-01

    Full Text Available Mining interesting patterns from DNA sequences is one of the most challenging tasks in bioinformatics and computational biology. Maximal contiguous frequent patterns are preferable for expressing the function and structure of DNA sequences and hence can capture the common data characteristics among related sequences. Biologists are interested in finding frequent orderly arrangements of motifs that are responsible for similar expression of a group of genes. In order to reduce mining time and complexity, however, most existing sequence mining algorithms either focus on finding short DNA sequences or require explicit specification of sequence lengths in advance. The challenge is to find longer sequences without specifying sequence lengths in advance. In this paper, we propose an efficient approach to mining maximal contiguous frequent patterns from large DNA sequence datasets. The experimental results show that our proposed approach is memory-efficient and mines maximal contiguous frequent patterns within a reasonable time.

  20. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis.

    Science.gov (United States)

    Matsuda, M; Tazumi, A; Kagawa, S; Sekizuka, T; Murayama, O; Moore, J E; Millar, B C

    2006-01-06

    At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

  1. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

    Directory of Open Access Journals (Sweden)

    Moore JE

    2006-01-01

    Full Text Available Abstract Background At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Results Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. Conclusion High sequence similarity (99.5% or more was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

  2. Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants.

    Science.gov (United States)

    Ayliffe, M A; Scott, N S; Timmis, J N

    1998-06-01

    A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.

  3. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...... mitochondrial genome of its close relative C. albicans. The complete sequence has implications for both mitochondrial DNA replication and the evolution of linear DNA genomes....

  4. Predicting promoter activities of primary human DNA sequences

    Science.gov (United States)

    Irie, Takuma; Park, Sung-Joon; Yamashita, Riu; Seki, Masahide; Yada, Tetsushi; Sugano, Sumio; Nakai, Kenta; Suzuki, Yutaka

    2011-01-01

    We developed a computer program that can predict the intrinsic promoter activities of primary human DNA sequences. We observed promoter activity using a quantitative luciferase assay and generated a prediction model using multiple linear regression. Our program achieved a prediction accuracy correlation coefficient of 0.87 between the predicted and observed promoter activities. We evaluated the prediction accuracy of the program using massive sequencing analysis of transcriptional start sites in vivo. We found that it is still difficult to predict transcript levels in a strictly quantitative manner in vivo; however, it was possible to select active promoters in a given cell from the other silent promoters. Using this program, we analyzed the transcriptional landscape of the entire human genome. We demonstrate that many human genomic regions have potential promoter activity, and the expression of some previously uncharacterized putatively non-protein-coding transcripts can be explained by our prediction model. Furthermore, we found that nucleosomes occasionally formed open chromatin structures with RNA polymerase II recruitment where the program predicted significant promoter activities, although no transcripts were observed. PMID:21486745

  5. Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c/sub 2/ gene

    Energy Technology Data Exchange (ETDEWEB)

    Donohue, T.J.; McEwan, A.G.; Kaplan, S.

    1986-11-01

    The Rhodobacter sphaeroides cytochrome c/sub 2/ functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (M/sub r/ 14,000), were used to identify and clone the cytochrome c/sub 2/ structural gene (cycA). DNA sequence analysis of the cycA gene indicated the presence of a typical procaryotic 21-residue signal sequence, suggesting that this periplasmic protein is synthesized in vivo as a precursor. Synthesis of an immunoreactive cytochrome c/sub 2/ precursor protein (M/sub r/ 15,500) was observed in vitro when plasmids containing the cycA gene were used as templates in an R. sphaeroides coupled transcription-translation system. Approximately 500 base pairs of DNA upstream of the cycA gene was sufficient to allow expression of this gene product in vitro. Northern blot analysis with an internal cycA-specific probe identified at least two possibly monocistronic transcripts present in both different cellular levels and relative stoichiometries in steady-state cells grown under different physiological conditions. The ratio of the small (740-mucleotide) and large (920-nucleotide) cycA-specific mRNA species was dependent on cultural conditions but was not affected by light intensity under photosynthetic conditions. These results suggest that the increase in the cellular level of the cytochrome c/sub 2/ protein found in photosynthetic cells was due, in part, to increased transcription of the single-copy cyc operon.

  6. Reversible DNA encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA 'fossils'.

    Science.gov (United States)

    Paunescu, Daniela; Puddu, Michela; Soellner, Justus O B; Stoessel, Philipp R; Grass, Robert N

    2013-12-01

    This protocol describes a method for encapsulating DNA into amorphous silica (glass) spheres, mimicking the protection of nucleic acids within ancient fossils. In this approach, DNA encapsulation is achieved after the ammonium functionalization of silica nanoparticles. Within the glass spheres, the nucleic acid molecules are hermetically sealed and protected from chemical attack, thereby withstanding high temperatures and aggressive radical oxygen species (ROS). The encapsulates can be used as inert taggants to trace chemical and biological entities. The present protocol is applicable to short double-stranded (ds) and single-stranded (ss) DNA fragments, genomic DNA and plasmids. The nucleic acids can be recovered from the glass spheres without harm by using fluoride-containing buffered oxide etch solutions. Special emphasis is placed in this protocol on the safe handling of these buffered hydrogen fluoride solutions. After dissolution of the spheres and subsequent purification, the nucleic acids can be analyzed by standard techniques (gel electrophoresis, quantitative PCR (qPCR) and sequencing). The protocol requires 6 d for completion with a total hands-on time of 4 h.

  7. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...... extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.......Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...

  8. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

    Science.gov (United States)

    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  9. Frequency and organization of papA homologous DNA sequences among uropathogenic digalactoside-binding Escherichia coli strains.

    OpenAIRE

    Denich, K; Craiu, A; Rugo, H; Muralidhar, G; O'Hanley, P

    1991-01-01

    The frequency of selected papA DNA sequences among 89 digalactoside-binding, uropathogenic Escherichia coli strains was evaluated with 12 different synthetic 15-base probes corresponding to papA genes from four digalactoside-binding piliated recombinant strains (HU849, 201B, and 200A). The papA probes encode amino acids which are common at the carboxy terminus of all strains, adjacent to the proximal portion of the intramolecular disulfide loop of strain 210B, or predicted to constitute the t...

  10. [Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

    Science.gov (United States)

    Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

    2017-08-01

    To analyze and detect the whole genome sequence of human mitochondrial DNA (mtDNA) by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

  11. True single-molecule DNA sequencing of a pleistocene horse bone

    Science.gov (United States)

    Orlando, Ludovic; Ginolhac, Aurelien; Raghavan, Maanasa; Vilstrup, Julia; Rasmussen, Morten; Magnussen, Kim; Steinmann, Kathleen E.; Kapranov, Philipp; Thompson, John F.; Zazula, Grant; Froese, Duane; Moltke, Ida; Shapiro, Beth; Hofreiter, Michael; Al-Rasheid, Khaled A.S.; Gilbert, M. Thomas P.; Willerslev, Eske

    2011-01-01

    Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first “true single molecule sequencing” of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3′ ends of ancient templates, indicating the presence of 3′-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches. PMID:21803858

  12. Rapid and affordable genome-wide bisulfite DNA sequencing by XmaI-reduced representation bisulfite sequencing.

    Science.gov (United States)

    Tanas, Alexander S; Borisova, Marina E; Kuznetsova, Ekaterina B; Rudenko, Viktoria V; Karandasheva, Kristina O; Nemtsova, Marina V; Izhevskaya, Vera L; Simonova, Olga A; Larin, Sergey S; Zaletaev, Dmitry V; Strelnikov, Vladimir V

    2017-06-01

    To develop a reduced representation bisulfite sequencing (RRBS) approach for rapid and affordable genome-wide DNA methylation analysis. We have selected restriction endonuclease XmaI to produce RRBS library fragments. After digestion and partial fill-in DNA fragments were ligated to barcoded adapters, bisulfite converted, size-selected, and sequenced on the Ion Torrent Personal Genome Machine. XmaI-RRBS results were compared with the previously published RRBS data. We have developed an XmaI-RRBS method for rapid and affordable genome-wide DNA methylation analysis, with library preparation taking only 4 days and sequencing possible within 4 h. We have also addressed several challenges in order to further improve the RRBS technology. XmaI-RRBS may be performed on degraded DNA samples and is compatible with the bench-top next-generation sequencing machines.

  13. The mitochondrial DNA sequence specificity of the anti-tumour drug bleomycin using end-labeled DNA and capillary electrophoresis and a comparison with genome-wide DNA sequencing.

    Science.gov (United States)

    Chung, Long H; Murray, Vincent

    2016-01-01

    The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was investigated in two human mitochondrial DNA sequences. Bleomycin was found to cleave preferentially at 5'-TGT*A-3' DNA sequences (where * is the cleavage site). The bleomycin analysis using capillary electrophoresis with laser-induced fluorescence was determined on both DNA strands and each strand was independently fluorescently labelled at the 3'- and 5'-ends. There was a high level of correlation between the intensity of bleomycin cleavage sites analysed by 3'- and 5'-end labelling. This is the first occasion that a comprehensive comparison has been made between these two end-labelling procedures to quantify cleavage by a DNA damaging agent and to investigate end-label bias. A comparison was also made between the bleomycin DNA sequence specificity obtained from genome-wide next-generation sequencing with that obtained from purified plasmid DNA sequences. This was accomplished by cloning sections of human mitochondrial DNA and comparing these identical mitochondrial DNA in the human mitochondrial genome. At individual sites, there was a very low level of correlation between bleomycin cleavage in plasmid sequencing and genome-wide sequencing. However, the overall bleomycin DNA sequence specificity was very similar in the two environments, namely 5'-TGT*A-3'. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  15. Microsatellite DNA in genomic survey sequences and UniGenes of loblolly pine

    Science.gov (United States)

    Craig S Echt; Surya Saha; Dennis L Deemer; C Dana Nelson

    2011-01-01

    Genomic DNA sequence databases are a potential and growing resource for simple sequence repeat (SSR) marker development in loblolly pine (Pinus taeda L.). Loblolly pine also has many expressed sequence tags (ESTs) available for microsatellite (SSR) marker development. We compared loblolly pine SSR densities in genome survey sequences (GSSs) to those in non-redundant...

  16. A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences.

    Directory of Open Access Journals (Sweden)

    David Caramelli

    Full Text Available BACKGROUND: DNA sequences from ancient specimens may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal and early modern (Cro-Magnoid Europeans. METHODOLOGY/PRINCIPAL FINDINGS: We typed the mitochondrial DNA (mtDNA hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23 and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. CONCLUSIONS/SIGNIFICANCE: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans.

  17. OPTSDNA: Performance evaluation of an efficient distributed bioinformatics system for DNA sequence analysis.

    Science.gov (United States)

    Khan, Mohammad Ibrahim; Sheel, Chotan

    2013-01-01

    Storage of sequence data is a big concern as the amount of data generated is exponential in nature at several locations. Therefore, there is a need to develop techniques to store data using compression algorithm. Here we describe optimal storage algorithm (OPTSDNA) for storing large amount of DNA sequences of varying length. This paper provides performance analysis of optimal storage algorithm (OPTSDNA) of a distributed bioinformatics computing system for analysis of DNA sequences. OPTSDNA algorithm is used for storing various sizes of DNA sequences into database. DNA sequences of different lengths were stored by using this algorithm. These input DNA sequences are varied in size from very small to very large. Storage size is calculated by this algorithm. Response time is also calculated in this work. The efficiency and performance of the algorithm is high (in size calculation with percentage) when compared with other known with sequential approach.

  18. A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer

    DEFF Research Database (Denmark)

    Álvarez-Martos, Isabel; Ferapontova, Elena

    2017-01-01

    A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding...... of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained...... by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus...

  19. Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma.

    Science.gov (United States)

    Warton, Kristina; Lin, Vita; Navin, Tina; Armstrong, Nicola J; Kaplan, Warren; Ying, Kevin; Gloss, Brian; Mangs, Helena; Nair, Shalima S; Hacker, Neville F; Sutherland, Robert L; Clark, Susan J; Samimi, Goli

    2014-06-15

    Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

  20. Protein and DNA sequence determinants of thermophilic adaptation.

    Directory of Open Access Journals (Sweden)

    Konstantin B Zeldovich

    2007-01-01

    Full Text Available There have been considerable attempts in the past to relate phenotypic trait--habitat temperature of organisms--to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from -10 degrees C to 110 degrees C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL, and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = -0.10. On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.

  1. Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

    Science.gov (United States)

    Wagatsuma, M; Hashimoto, K; Matsukura, T

    1990-01-01

    We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed. Images PMID:2153245

  2. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  3. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, J; Gordon, L A; Olsen, A; Terry, A; Schmutz, J; Lamerdin, J; Hellsten, U; Goodstein, D; Couronne, O; Tran-Gyamfi, M

    2004-04-06

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high GC content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in Mendelian disorders, including familial hypercholesterolemia and insulin-resistant diabetes. Nearly one quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  4. [Patentability of DNA sequences: the debate remains open].

    Science.gov (United States)

    Martín Uranga, Amelia

    2013-01-01

    The patentability of human genes was from the beginning of the discussion concerning the Directive on the legal protection of biotechnological inventions, an issue that provoked debates among politicians, scientists, lawyers and civil society itself. Although Directive 98/44 tried to settle the matter by stating that to support the patentability of human genes, it should know what role they fulfill, which protein they encode, all of this as an essential requirement to test its industrial application. However, following the judgment of 13 June 2013 (Supreme Court of the United States of America in the case of Association for Molecular Pathology et al. versus Myriad Genetics Inc.) the debate on this issue has been reopened. There are several issues to be considered, taking into account that the patents on DNA & Gene Sequences have played an important incentive to increase the interest in biotechnology applied to human health. On the other hand, this is a paradigm shift in the R & D of biopharmaceutical companies, and it has moved from an in house research model to a model of open innovation, a model of collaboration between large corporations with biotech SMEs and public and private research centers. This model of innovation, impacts on the issue of the industrial property, and therefore it will be necessary to clearly define what each party brings to the relationship and how they are expected to share the results. But all of this, with the ultimate goal that the patients have access to treatments and medications most innovative, safe and effective.

  5. Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

    DEFF Research Database (Denmark)

    Binladen, Jonas; Wiuf, Carsten Henrik; Gilbert, M. Thomas P.

    2006-01-01

    in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from...

  6. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

  7. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the cathepsin B like proteases, suggesting that HC58cDNA was a member of the papain family. Keywords:Haemonchus contortus, HC58cDNA, cathepsin B like protease, papain family. Kenya Veterinarian Vol.

  8. Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

    NARCIS (Netherlands)

    Kuipers, A.G.J.; Kamstra, S.A.; Jeu, de M.J.; Jacobsen, E.

    2002-01-01

    Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A

  9. Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K.; Bradbury, E.M.; Garcia, A.E.

    1997-07-01

    This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.

  10. HPLC analysis of 4',5'-monoadduct formation in calf thymus DNA and synthetic polynucleotides treated with UVA and 8-methoxypsoralen

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Bagel, J.; Edelson, R.L.

    1985-01-01

    8-methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8-MOP at each of the irradiating wavelengths showed that the 4',5'-furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8-MOP photoreactivity: (poly(dAdT.dAdT)>poly(dA.dT)>poly(dGdC.dGdC) in both the B and Z forms>poly(dT). (author)

  11. A Microbiome DNA Enrichment Method for Next-Generation Sequencing Sample Preparation.

    Science.gov (United States)

    Yigit, Erbay; Feehery, George R; Langhorst, Bradley W; Stewart, Fiona J; Dimalanta, Eileen T; Pradhan, Sriharsa; Slatko, Barton; Gardner, Andrew F; McFarland, James; Sumner, Christine; Davis, Theodore B

    2016-07-01

    "Microbiome" is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next-generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  12. Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing.

    Science.gov (United States)

    Davis, Carey; Peters, Dixie; Warshauer, David; King, Jonathan; Budowle, Bruce

    2015-03-01

    Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

    Science.gov (United States)

    Mayjonade, Baptiste; Gouzy, Jérôme; Donnadieu, Cécile; Pouilly, Nicolas; Marande, William; Callot, Caroline; Langlade, Nicolas; Muños, Stéphane

    2016-10-01

    De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.

  14. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    Science.gov (United States)

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

  15. Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence

    Science.gov (United States)

    Takizawa, Yoshimasa; Tanaka, Hiroki; Machida, Shinichi; Koyama, Masako; Maehara, Kazumitsu; Wade, Paul A.; Wolf, Matthias

    2018-01-01

    Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA. PMID:29563192

  16. Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae allotetraploids

    Directory of Open Access Journals (Sweden)

    Soltis Pamela S

    2010-09-01

    Full Text Available Abstract Background Tragopogon mirus and T. miscellus are allotetraploids (2n = 24 that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12 from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis. We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. Results Using Southern blot hybridization and fluorescent in situ hybridization (FISH, we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals and four lines of synthetic T. miscellus (71 individuals. Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. Conclusions Uniparental reductions of

  17. How effective is graphene nanopore geometry on DNA sequencing?

    OpenAIRE

    Satarifard, Vahid; Foroutan, Masumeh; Ejtehadi, Mohammad Reza

    2015-01-01

    In this paper we investigate the effects of graphene nanopore geometry on homopolymer ssDNA pulling process through nanopore using steered molecular dynamic (SMD) simulations. Different graphene nanopores are examined including axially symmetric and asymmetric monolayer graphene nanopores as well as five layer graphene polyhedral crystals (GPC). The pulling force profile, moving fashion of ssDNA, work done in irreversible DNA pulling and orientations of DNA bases near the nanopore are assesse...

  18. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...... extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature....

  19. Improving the performance of true single molecule sequencing for ancient DNA

    Science.gov (United States)

    2012-01-01

    Background Second-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase. Results We found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted. Conclusions We propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted. PMID:22574620

  20. Sequence analysis of mitochondrial DNA hypervariable region III of ...

    African Journals Online (AJOL)

    The aims of this research were to study mitochondrial DNA hypervariable region III and establish the degree of variation characteristic of a fragment. The mitochondrial DNA (mtDNA) is a small circular genome located within the mitochondria in the cytoplasm of the cell and a smaller 1.2 kb pair fragment, called the control ...

  1. Agarose Gel Size Selection for DNA Sequencing Libraries.

    Science.gov (United States)

    Mardis, Elaine; McCombie, W Richard

    2017-08-01

    Agarose gel electrophoresis may be used to purify fragmented genomic DNA after ligation of adaptors. After electrophoresis, the region of the gel containing the desired size range of DNA is excised, and the DNA is subsequently extracted from the gel and purified by passage through a spin column. © 2017 Cold Spring Harbor Laboratory Press.

  2. Sequence analysis of mitochondrial DNA hypervariable region III of ...

    African Journals Online (AJOL)

    Aghomotsegin

    2015-07-01

    Jul 1, 2015 ... may in time accumulate differences in the mitochondrial. DNA but show little difference in the nuclear DNA and finally, maternal inheritance: A further reason for the use of mitochondrial DNA in species testing, and in forensic science, is its mode of inheritance. Mitochondria exist within the cytoplasm of cells, ...

  3. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

    Science.gov (United States)

    Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

    2015-01-01

    The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

  4. Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

    Science.gov (United States)

    Rackwitz, Jenny; Bald, Ilko

    2018-03-26

    During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification.

    Science.gov (United States)

    Bassaganyas, Laia; Freedman, George; Vaka, Dedeepya; Wan, Eunice; Lao, Richard; Chen, Flavia; Kvale, Mark; Currier, Robert J; Puck, Jennifer M; Kwok, Pui-Yan

    2018-01-01

    Newborn screening (NBS) for rare conditions is performed in all 50 states in the USA. We have partnered with the California Department of Public Health Genetic Disease Laboratory to determine whether sufficient DNA can be extracted from archived dried blood spots (DBS) for next-generation sequencing in the hopes that next-generation sequencing can play a role in NBS. We optimized the DNA extraction and sequencing library preparation protocols for residual infant DBS archived over 20 years ago and successfully obtained acceptable whole exome and whole genome sequencing data. This sequencing study using DBS DNA without whole genome amplification prior to sequencing library preparation provides evidence that properly stored residual newborn DBS are a satisfactory source of DNA for genetic studies. © 2017 Wiley Periodicals, Inc.

  6. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  7. Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples.

    OpenAIRE

    Chen, D; Harke, H R; Dovichi, N J

    1992-01-01

    We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate...

  8. Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA.

    Science.gov (United States)

    Sproul, John S; Maddison, David R

    2017-11-01

    Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10 ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens. © 2017 John Wiley & Sons Ltd.

  9. eDNA Barcoding: Using Next-Generation Sequencing of Environmental DNA for Detection and Identification of Cetacean Species

    Science.gov (United States)

    2015-09-30

    mmi.oregonstate.edu/ccgl LONG - TERM GOALS We are developing next-generation sequencing and digital (d)PCR methodology for detection and species...ubiquitous DNA sequencing for surveys of biodiversity more efficient and affordable in the near future. RELATED PROJECTS None to date.

  10. Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines

    Directory of Open Access Journals (Sweden)

    Joanne G. Bartlett

    2014-01-01

    Full Text Available Sequencing across the junction between an integrated transfer DNA (T-DNA and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression.

  11. Two dimensional molecular electronics spectroscopy for molecular fingerprinting, DNA sequencing, and cancerous DNA recognition.

    Science.gov (United States)

    Rajan, Arunkumar Chitteth; Rezapour, Mohammad Reza; Yun, Jeonghun; Cho, Yeonchoo; Cho, Woo Jong; Min, Seung Kyu; Lee, Geunsik; Kim, Kwang S

    2014-02-25

    Laser-driven molecular spectroscopy of low spatial resolution is widely used, while electronic current-driven molecular spectroscopy of atomic scale resolution has been limited because currents provide only minimal information. However, electron transmission of a graphene nanoribbon on which a molecule is adsorbed shows molecular fingerprints of Fano resonances, i.e., characteristic features of frontier orbitals and conformations of physisorbed molecules. Utilizing these resonance profiles, here we demonstrate two-dimensional molecular electronics spectroscopy (2D MES). The differential conductance with respect to bias and gate voltages not only distinguishes different types of nucleobases for DNA sequencing but also recognizes methylated nucleobases which could be related to cancerous cell growth. This 2D MES could open an exciting field to recognize single molecule signatures at atomic resolution. The advantages of the 2D MES over the one-dimensional (1D) current analysis can be comparable to those of 2D NMR over 1D NMR analysis.

  12. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  13. Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

    Science.gov (United States)

    M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

    2009-01-01

    The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

  14. Recent applications of DNA sequencing technologies in food, nutrition and agriculture.

    Science.gov (United States)

    Liu, George E

    2011-09-01

    Next-generation DNA sequencing technologies are able to produce millions of short sequence reads in a highthroughput, cost-effective fashion. The emergence of these technologies has not only facilitated genome sequencing but also changed the landscape of life sciences. This review surveys their recent applications in food, nutrition and agriculture ranging from whole-genome sequencing and resequencing, RNA-seq and ChIP-seq, structural, functional and comparative genomics to metagenomics and epigenetics. We already began to witness broad impacts of these DNA sequencing technologies for solving the complex biological problems in food, nutrition and agriculture. In this article, recent patent-based information is also included.

  15. Recent Applications of DNA Sequencing Technologies in Food, Nutrition and Agriculture

    Science.gov (United States)

    Next-generation DNA sequencing technologies are able to produce millions of short sequence reads in a high-throughput, cost-effective fashion. The emergence of these technologies has not only facilitated genome sequencing but also changed the landscape of life sciences. This review surveys their rec...

  16. Numerical encoding of DNA sequences by chaos game representation with application in similarity comparison.

    Science.gov (United States)

    Hoang, Tung; Yin, Changchuan; Yau, Stephen S-T

    2016-10-01

    Numerical encoding plays an important role in DNA sequence analysis via computational methods, in which numerical values are associated with corresponding symbolic characters. After numerical representation, digital signal processing methods can be exploited to analyze DNA sequences. To reflect the biological properties of the original sequence, it is vital that the representation is one-to-one. Chaos Game Representation (CGR) is an iterative mapping technique that assigns each nucleotide in a DNA sequence to a respective position on the plane that allows the depiction of the DNA sequence in the form of image. Using CGR, a biological sequence can be transformed one-to-one to a numerical sequence that preserves the main features of the original sequence. In this research, we propose to encode DNA sequences by considering 2D CGR coordinates as complex numbers, and apply digital signal processing methods to analyze their evolutionary relationship. Computational experiments indicate that this approach gives comparable results to the state-of-the-art multiple sequence alignment method, Clustal Omega, and is significantly faster. The MATLAB code for our method can be accessed from: www.mathworks.com/matlabcentral/fileexchange/57152. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Production of Epitope-Specific Antibodies by Immunization with Synthetic Epitope Peptide Formulated with CpG-DNA-Liposome Complex Without Carriers.

    Science.gov (United States)

    Kim, Dongbum; Lee, Younghee; Kwon, Hyung-Joo

    2015-01-01

    Antibody production using synthetic peptides has been investigated extensively to develop therapeutic antibodies and prophylactic vaccines. Previously, we reported that a complex of CpG-DNA and synthetic peptides corresponding to B cell epitopes, encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex, significantly enhanced the synthetic peptide-specific IgG production. Here, we describe synthetic peptide-based epitope screening and antibody production without conventional carriers.

  18. The impact of DNA input amount and DNA source on the performance of whole-exome sequencing in cancer epidemiology.

    Science.gov (United States)

    Zhu, Qianqian; Hu, Qiang; Shepherd, Lori; Wang, Jianmin; Wei, Lei; Morrison, Carl D; Conroy, Jeffrey M; Glenn, Sean T; Davis, Warren; Kwan, Marilyn L; Ergas, Isaac J; Roh, Janise M; Kushi, Lawrence H; Ambrosone, Christine B; Liu, Song; Yao, Song

    2015-08-01

    Whole-exome sequencing (WES) has recently emerged as an appealing approach to systematically study coding variants. However, the requirement for a large amount of high-quality DNA poses a barrier that may limit its application in large cancer epidemiologic studies. We evaluated the performance of WES with low input amount and saliva DNA as an alternative source material. Five breast cancer patients were randomly selected from the Pathways Study. From each patient, four samples, including 3 μg, 1 μg, and 0.2 μg blood DNA and 1 μg saliva DNA, were aliquoted for library preparation using the Agilent SureSelect Kit and sequencing using Illumina HiSeq2500. Quality metrics of sequencing and variant calling, as well as concordance of variant calls from the whole exome and 21 known breast cancer genes, were assessed by input amount and DNA source. There was little difference by input amount or DNA source on the quality of sequencing and variant calling. The concordance rate was about 98% for single-nucleotide variant calls and 83% to 86% for short insertion/deletion calls. For the 21 known breast cancer genes, WES based on low input amount and saliva DNA identified the same set variants in samples from a same patient. Low DNA input amount, as well as saliva DNA, can be used to generate WES data of satisfactory quality. Our findings support the expansion of WES applications in cancer epidemiologic studies where only low DNA amount or saliva samples are available. ©2015 American Association for Cancer Research.

  19. Bacterial promoter prediction: Selection of dynamic and static physical properties of DNA for reliable sequence classification.

    Science.gov (United States)

    Ryasik, Artem; Orlov, Mikhail; Zykova, Evgenia; Ermak, Timofei; Sorokin, Anatoly

    2018-01-30

    Predicting promoter activity of DNA fragment is an important task for computational biology. Approaches using physical properties of DNA to predict bacterial promoters have recently gained a lot of attention. To select an adequate set of physical properties for training a classifier, various characteristics of DNA molecule should be taken into consideration. Here, we present a systematic approach that allows us to select less correlated properties for classification by means of both correlation and cophenetic coefficients as well as concordance matrices. To prove this concept, we have developed the first classifier that uses not only sequence and static physical properties of DNA fragment, but also dynamic properties of DNA open states. Therefore, the best performing models with accuracy values up to 90% for all types of sequences were obtained. Furthermore, we have demonstrated that the classifier can serve as a reliable tool enabling promoter DNA fragments to be distinguished from promoter islands despite the similarity of their nucleotide sequences.

  20. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

    DEFF Research Database (Denmark)

    Tornøe, Jens; Kusk, P.; Johansen, T.E.

    2002-01-01

    The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C...

  1. Close sequence identity between ribosomal DNA episomes of the ...

    Indian Academy of Sciences (India)

    In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1.6% in the two species. The most divergent sequence between E. histolytica and E. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS2 ...

  2. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Pongo abelii, Macaca fascicularis, Mus musculus, Bos taurus and Rattus norvegicus are 93.1, 92.5, 92.2, 91.1, 90.6 and 90.0% respectively. The amino acid sequence encoded by RPS20 ...

  3. Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Michael V Zaragoza

    2010-08-01

    Full Text Available Mutations in mitochondrial DNA (mtDNA may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300x average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20x while heteroplasmic variants required >200x coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

  4. An Automated Sample Preparation System for Large-Scale DNA Sequencing

    Science.gov (United States)

    Marziali, Andre; Willis, Thomas D.; Federspiel, Nancy A.; Davis, Ronald W.

    1999-01-01

    Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates. PMID:10330125

  5. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  6. Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

    Directory of Open Access Journals (Sweden)

    Oliver Hawlitschek

    2011-02-01

    Full Text Available DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data.The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n.In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

  7. Synthetic approaches to nucleopeptides containing all four nucleobases, and nucleic acid-binding studies on a mixed-sequence nucleo-oligolysine.

    Science.gov (United States)

    Roviello, Giovanni N; Musumeci, Domenica

    2016-07-16

    In this article we describe two solid-phase synthetic routes to obtain a nucleo-oligolysine α-peptide containing all four natural nucleobases. The first one is based on the oligomerization of the nucleobase-containing monomers, easily synthesized as herein described. The second strategy has the advantage of avoiding the solution synthesis of the monomeric building blocks, leading to the final nucleopeptide by direct solid-phase couplings of the suitably protected nucleobases with the free amino groups on the growing peptide chain still anchored to the resin. Both strategies are general and can be applied to the synthesis of nucleopeptides having backbones formed by any other diamino acid moiety decorated with the four nucleobases. We also report the CD and UV studies on the hybridization properties of the obtained nucleopeptide, containing all four nucleobases on alternate lysines in the sequence, towards complementary DNA and RNA strands. The nucleo-oligolysine with a mixed-base sequence did not prove to bind complementary DNA, but was able to recognize the complementary RNA forming a complex with a higher melting temperature than that of the corresponding RNA/RNA natural duplex and comparable with that of the analogous PNA/RNA system.

  8. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation.

    Directory of Open Access Journals (Sweden)

    Si-Yang Liu

    Full Text Available Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.

  9. High-speed automated DNA sequencing utilizing from-the-side laser excitation

    Science.gov (United States)

    Westphall, Michael S.; Brumley, Robert L., Jr.; Buxton, Erin C.; Smith, Lloyd M.

    1995-04-01

    The Human Genome Initiative is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. If successfully completed, the resultant sequence database will be a tool of unparalleled power for biomedical research. One of the major challenges of this project is in the area of DNA sequencing technology. At this time, virtually all DNA sequencing is based upon the separation of DNA fragments in high resolution polyacrylamide gels. This method, as generally practiced, is one to two orders of magnitude too slow and expensive for the successful completion of the Human Genome projection. One reasonable approach is improved sequencing of DNA fragments is to increase the performance of such gel-based sequencing methods. Decreased sequencing times may be obtained by increasing the magnitude of the electric field employed. This is not possible with conventional sequencing, due to the fact that the additional heat associated with the increased electric field cannot be adequately dissipated. Recent developments in the use of thin gels have addressed this problem. Performing electrophoresis in ultrathin (50 to 100 microns) gels greatly increases the heat transfer efficiency, thus allowing the benefits of larger electric fields to be obtained. An increase in separation speed of about an order of magnitude is readily achieved. Thin gels have successfully been used in capillary and slab formats. A detection system has been designed for use with a multiple fluorophore sequencing strategy in horizontal ultrathin slab gels. The system employs laser through-the-side excitation and a cooled CCD detector; this allows for the parallel detection of up to 24 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (115 micrometers ) denaturing polyacrylamide gels. Four hundred bases of sequence information is obtained from 100 ng of M13 template DNA in an hour, corresponding to an

  10. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

    Science.gov (United States)

    Fan, H. Christina; Blumenfeld, Yair J.; Chitkara, Usha; Hudgins, Louanne; Quake, Stephen R.

    2008-01-01

    We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes. PMID:18838674

  11. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  12. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  13. Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis.

    Science.gov (United States)

    Goldfeder, Rachel L; Wall, Dennis P; Khoury, Muin J; Ioannidis, John P A; Ashley, Euan A

    2017-10-15

    Most human diseases have underlying genetic causes. To better understand the impact of genes on disease and its implications for medicine and public health, researchers have pursued methods for determining the sequences of individual genes, then all genes, and now complete human genomes. Massively parallel high-throughput sequencing technology, where DNA is sheared into smaller pieces, sequenced, and then computationally reordered and analyzed, enables fast and affordable sequencing of full human genomes. As the price of sequencing continues to decline, more and more individuals are having their genomes sequenced. This may facilitate better population-level disease subtyping and characterization, as well as individual-level diagnosis and personalized treatment and prevention plans. In this review, we describe several massively parallel high-throughput DNA sequencing technologies and their associated strengths, limitations, and error modes, with a focus on applications in epidemiologic research and precision medicine. We detail the methods used to computationally process and interpret sequence data to inform medical or preventative action. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Characterization of the complete nuclear ribosomal DNA sequences of Eurytrema pancreaticum.

    Science.gov (United States)

    Su, X; Zhang, Y; Zheng, X; Wang, X X; Li, Y; Li, Q; Wang, C R

    2017-06-27

    Eurytrema pancreaticum is one of the most common trematodes of cattle and sheep, and also infects humans occasionally, causing great economic losses and medical costs. In this study, the sequences of the complete nuclear ribosomal DNA (rDNA) repeat units of five E. pancreaticum individuals were determined for the first time. They were 8306-8310 bp in length, including the small subunit (18S) rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), large subunit (28S) rDNA and intergenic spacer (IGS). There were no length variations in any of the investigated 18S (1996 bp), ITS1 (1103 bp), 5.8S (160 bp), ITS2 (231 bp) or 28S (3669 bp) rDNA sequences, whereas the IGS rDNA sequences of E. pancreaticum had a 4-bp length variation, ranging from 1147 to 1151 bp. The intraspecific variations within E. pancreaticum were 0-0.2% for 18S rDNA, 0-0.5% for ITS1, 0% for 5.8S rDNA and ITS2, 0-0.2% for 28S rDNA and 2.9-20.2% for IGS. There were nine types of repeat sequences in ITS1, two types in 28S rDNA, but none in IGS. A phylogenetic analysis based on the 18S rDNA sequences classified E. pancreaticum in the family Dicrocoeliidae of Plagiorchiata, closely related to the suborder Opisthorchiata. These results provide useful information for the further study of Dicrocoeliidae trematodes.

  15. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud.

    Science.gov (United States)

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-07-08

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Application of Quaternion in improving the quality of global sequence alignment scores for an ambiguous sequence target in Streptococcus pneumoniae DNA

    Science.gov (United States)

    Lestari, D.; Bustamam, A.; Novianti, T.; Ardaneswari, G.

    2017-07-01

    DNA sequence can be defined as a succession of letters, representing the order of nucleotides within DNA, using a permutation of four DNA base codes including adenine (A), guanine (G), cytosine (C), and thymine (T). The precise code of the sequences is determined using DNA sequencing methods and technologies, which have been developed since the 1970s and currently become highly developed, advanced and highly throughput sequencing technologies. So far, DNA sequencing has greatly accelerated biological and medical research and discovery. However, in some cases DNA sequencing could produce any ambiguous and not clear enough sequencing results that make them quite difficult to be determined whether these codes are A, T, G, or C. To solve these problems, in this study we can introduce other representation of DNA codes namely Quaternion Q = (PA, PT, PG, PC), where PA, PT, PG, PC are the probability of A, T, G, C bases that could appear in Q and PA + PT + PG + PC = 1. Furthermore, using Quaternion representations we are able to construct the improved scoring matrix for global sequence alignment processes, by applying a dot product method. Moreover, this scoring matrix produces better and higher quality of the match and mismatch score between two DNA base codes. In implementation, we applied the Needleman-Wunsch global sequence alignment algorithm using Octave, to analyze our target sequence which contains some ambiguous sequence data. The subject sequences are the DNA sequences of Streptococcus pneumoniae families obtained from the Genebank, meanwhile the target DNA sequence are received from our collaborator database. As the results we found the Quaternion representations improve the quality of the sequence alignment score and we can conclude that DNA sequence target has maximum similarity with Streptococcus pneumoniae.

  17. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

    Directory of Open Access Journals (Sweden)

    Soichi Inagaki

    Full Text Available Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  18. DNA polymorphisms in the Sahiwal breed of Zebu cattle revealed by synthetic oligonucleotide probes

    International Nuclear Information System (INIS)

    Shashikanth; Yadav, B.R.

    2005-01-01

    Genomic DNA of 15 randomly selected unrelated animals and from two sire families (11 animals) of the Sahiwal breed of Zebu cattle were investigated. Four oligonucleotide probes - (GTG) 5 , (TCC) 5 , (GT) 8 and (GT) 12 - were used on genomic DNA digested with restriction enzymes AluI, HinfI, MboI, EcoRI and HaeIII in different combinations. All four probes produced multiloci fingerprints with differing levels of polymorphisms. Total bands and shared bands in the fingerprints of each individual were in the range of 2.5 to 23.0 KB. Band number ranged from 9 to 17, with 0.48 average band sharing. Probes (GT) 8 , (GT) 12 and (TCC) 5 produced fingerprinting patterns of medium to low polymorphism, whereas probe (GTG) 5 produced highly polymorphic patterns. Probe (GTG) 5 in combination with the HaeIII enzyme was highly polymorphic with a heterozygosity level of 0.85, followed by (GT) 8 , (TCC) 5 and (GT) 12 with heterozygosity levels of 0.70, 0.65 and 0.30, respectively. Probe GTG 5 or its complementary sequence CAC 5 produced highly polymorphic fingerprints, indicating that the probe can be used for analysing population structure, parentage verification and identifying loci controlling quantitative traits and fertility status. (author)

  19. Screening of selected starter cultures for the presence of DNA sequences coding for tyrosine decarboxylase

    Directory of Open Access Journals (Sweden)

    Radka Burdychová

    2006-01-01

    Full Text Available Here, seven different starter cultures used in the production of fermented sausages were screened for the presence or absence of specific DNA sequences coding for tyrosine decarboxylase. PCR with the a set of specific primers TDC2/TDC5 (COTON et al., 2004 was used. The PCR analysis of DNA from two starter cultures confirmed the presence of DNA sequences for tyrosine decarboxylase. A detailed analysis of the starter cultures showed that DNA sequences for tyrosine decarboxylase are contained in genomic DNA of Lactobacillus curvatus and Lactobacillus sakei. These results show suitability of the described PCR method for the screening of starter cultures for the presence of the gene for tyrosine decarboxylase that is responsible for the production of the biogenic amine tyramine.

  20. Plant DNA sequences from feces: potential means for assessing diets of wild primates.

    Science.gov (United States)

    Bradley, Brenda J; Stiller, Mathias; Doran-Sheehy, Diane M; Harris, Tara; Chapman, Colin A; Vigilant, Linda; Poinar, Hendrik

    2007-06-01

    Analyses of plant DNA in feces provides a promising, yet largely unexplored, means of documenting the diets of elusive primates. Here we demonstrate the promise and pitfalls of this approach using DNA extracted from fecal samples of wild western gorillas (Gorilla gorilla) and black and white colobus monkeys (Colobus guereza). From these DNA extracts we amplified, cloned, and sequenced small segments of chloroplast DNA (part of the rbcL gene) and plant nuclear DNA (ITS-2). The obtained sequences were compared to sequences generated from known plant samples and to those in GenBank to identify plant taxa in the feces. With further optimization, this method could provide a basic evaluation of minimum primate dietary diversity even when knowledge of local flora is limited. This approach may find application in studies characterizing the diets of poorly-known, unhabituated primate species or assaying consumer-resource relationships in an ecosystem. (c) 2007 Wiley-Liss, Inc.

  1. Multi-modulus algorithm based on global artificial fish swarm intelligent optimization of DNA encoding sequences.

    Science.gov (United States)

    Guo, Y C; Wang, H; Wu, H P; Zhang, M Q

    2015-12-21

    Aimed to address the defects of the large mean square error (MSE), and the slow convergence speed in equalizing the multi-modulus signals of the constant modulus algorithm (CMA), a multi-modulus algorithm (MMA) based on global artificial fish swarm (GAFS) intelligent optimization of DNA encoding sequences (GAFS-DNA-MMA) was proposed. To improve the convergence rate and reduce the MSE, this proposed algorithm adopted an encoding method based on DNA nucleotide chains to provide a possible solution to the problem. Furthermore, the GAFS algorithm, with its fast convergence and global search ability, was used to find the best sequence. The real and imaginary parts of the initial optimal weight vector of MMA were obtained through DNA coding of the best sequence. The simulation results show that the proposed algorithm has a faster convergence speed and smaller MSE in comparison with the CMA, the MMA, and the AFS-DNA-MMA.

  2. Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing

    DEFF Research Database (Denmark)

    Gamba, Cristina; Hanghøj, Kristian Ebbesen; Gaunitz, Charleen

    2016-01-01

    The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs...... of high-throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning...... a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules...

  3. A Short Review on the Synthetic Strategies of Duocarmycin Analogs that are Powerful DNA Alkylating Agents.

    Science.gov (United States)

    Patil, Pravin C; Satam, Vijay; Lee, Moses

    2015-01-01

    The duocarmycins and CC-1065 are members of a class of DNA minor groove, AT-sequence selective, and adenine-N3 alkylating agents, isolated from Streptomyces sp. that exhibit extremely potent cytotoxicity against the growth of cancer cells grown in culture. Initial synthesis and structural modification of the cyclopropa[c] pyrrolo[3,2-e]indole (CPI) DNA-alkylating motif as well as the indole non-covalent binding region in the 1980s have led to several compounds that entered clinical trials as potential anticancer drugs. However, due to significant systemic toxicity none of the analogs have passed clinical evaluation. As a result, the intensity in the design, synthesis, and development of novel analogs of the duocarmycins has continued. Accordingly, in this review, which covers a period from the 1990s through the present time, the design and synthesis of duocarmycin SA are described along with the synthesis of novel and highly cytotoxic analogs that lack the chiral center. Examples of achiral analogs of duocarmycin SA described in this review include seco-DUMSA (39 and 40), seco-amino-CBI-TMI (13, Centanamycin), and seco-hydroxy-CBI-TMI (14). In addition, another novel class of biologically active duocarmycin SA analogs that contained the seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) DNA alkylating submit was also designed and synthesized. The synthesis of seco-iso-CFI-TMI (10, Tafuramycin A) and seco-CFQ-TMI (11, Tafuramycin B) is included in this review.

  4. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality... scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  5. Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

    Science.gov (United States)

    Sasaki, Y; Takikawa, N; Kojima, A; Norimatsu, M; Suzuki, S; Tamura, Y

    2001-05-01

    The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

  6. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    Science.gov (United States)

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-10-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

  7. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    length τ-crystallin cDNA from crocodilian lens and α-enolase from other tissues. ... human (Acc. No. NM_001428). The sequences were used to construct a phylogenetic tree depicting gene lineage, using the clustering program DNAML.

  8. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  9. Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

    Science.gov (United States)

    Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

    2014-01-01

    Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

  10. Tsukamurella tyrosinosolvens intravascular catheter infection identified using 16S ribosomal DNA sequencing.

    Science.gov (United States)

    Sheridan, Elizabeth A S; Warwick, Simon; Chan, Anthony; Dall'Antonia, Martino; Koliou, Maria; Sefton, Armine

    2003-03-01

    Cultures of blood from a hemodialysis line repeatedly yielded a gram-positive rod. The organism was identified as Tsukamurella tyrosinosolvens by 16S ribosomal DNA sequencing, and the patient was treated successfully by removal of the line.

  11. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin.

    Science.gov (United States)

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-10-13

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecule, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G • U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G • U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

  12. Efficiency of ITS sequences for DNA barcoding in Passiflora (Passifloraceae).

    Science.gov (United States)

    Giudicelli, Giovanna Câmara; Mäder, Geraldo; de Freitas, Loreta Brandão

    2015-04-01

    DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using "best match" and "best close match" methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1) region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species.

  13. Efficiency of ITS Sequences for DNA Barcoding in Passiflora (Passifloraceae

    Directory of Open Access Journals (Sweden)

    Giovanna Câmara Giudicelli

    2015-04-01

    Full Text Available DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using “best match” and “best close match” methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1 region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species.

  14. Algorithms for mapping high-throughput DNA sequences

    DEFF Research Database (Denmark)

    Frellsen, Jes; Menzel, Peter; Krogh, Anders

    2014-01-01

    of data generation, new bioinformatics approaches have been developed to cope with the large amount of sequencing reads obtained in these experiments. In this chapter, we first introduce HTS technologies and their usage in molecular biology and discuss the problem of mapping sequencing reads...

  15. Solving the Curriculum Sequencing Problem with DNA Computing Approach

    Science.gov (United States)

    Debbah, Amina; Ben Ali, Yamina Mohamed

    2014-01-01

    In the e-learning systems, a learning path is known as a sequence of learning materials linked to each others to help learners achieving their learning goals. As it is impossible to have the same learning path that suits different learners, the Curriculum Sequencing problem (CS) consists of the generation of a personalized learning path for each…

  16. Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Science.gov (United States)

    Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

  17. A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures.

    Science.gov (United States)

    Bertolini, Francesca; Ghionda, Marco Ciro; D'Alessandro, Enrico; Geraci, Claudia; Chiofalo, Vincenzo; Fontanesi, Luca

    2015-01-01

    The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

  18. Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

    Science.gov (United States)

    Sharma, Aashish; Pohane, Amol Arunrao; Bansal, Sandhya; Bajaj, Avinash; Jain, Vikas; Srivastava, Aasheesh

    2015-02-23

    Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 μg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Multifractal analysis of DNA sequences using a novel chaos-game representation

    Science.gov (United States)

    Gutiérrez, J. M.; Rodríguez, M. A.; Abramson, G.

    2001-11-01

    We present a generalization of the standard chaos-game representation method introduced by Jeffrey. To this aim, a DNA symbolic sequence is mapped onto a singular measure on the attractor of a particular IFS model, which is a perfect statistical representation of the sequence. A multifractal analysis of the resulting measure is introduced and an interpretation of singularities in terms of mutual information and redundancy (statistical dependence) among subsequence symbols within the DNA sequence is provided. The multifractal spectrum is also shown to be more sensitive for detecting dependence structures within the DNA sequence than the averaged contribution given by redundancy. This method presents several advantages with respect to other representations such as walks or interfaces, which may introduce spurious effects. In contrast with the results obtained by other standard methods, here we note that no general statement can be made on the influence of coding and non-coding content on the correlation length of a given sequence.

  20. ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers.

    Science.gov (United States)

    Sorge, J A; Blinderman, L A

    1989-01-01

    A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, lambda phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating high-quality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing. Images PMID:2556705

  1. Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

    Science.gov (United States)

    Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

  2. Generating Multiple Base-Resolution DNA Methylomes Using Reduced Representation Bisulfite Sequencing.

    Science.gov (United States)

    Chatterjee, Aniruddha; Rodger, Euan J; Stockwell, Peter A; Le Mée, Gwenn; Morison, Ian M

    2017-01-01

    Reduced representation bisulfite sequencing (RRBS) is an effective technique for profiling genome-wide DNA methylation patterns in eukaryotes. RRBS couples size selection, bisulfite conversion, and second-generation sequencing to enrich for CpG-dense regions of the genome. The progressive improvement of second-generation sequencing technologies and reduction in cost provided an opportunity to examine the DNA methylation patterns of multiple genomes. Here, we describe a protocol for sequencing multiple RRBS libraries in a single sequencing reaction to generate base-resolution methylomes. Furthermore, we provide a brief guideline for base-calling and data analysis of multiplexed RRBS libraries. These strategies will be useful to perform large-scale, genome-wide DNA methylation analysis.

  3. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  4. Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

    Science.gov (United States)

    Soto, J; Bessodes, M; Pitard, B; Mailhe, P; Scherman, D; Byk, G

    2000-05-01

    We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

  5. Comparative d2/d3 LSU–rDNA sequence study of some Iranian ...

    African Journals Online (AJOL)

    SERVER

    2007-11-05

    Nov 5, 2007 ... as Serilanka and Japan (Sivapalan et al., 1986). It causes a sever decline of tea shrubs where it ... facility diagnosis of potential pest problems as well as improving prediction about pathogenicity and ... sequence analysis of coding and non-coding region of nuclear ribosomal DNA (rDNA) have became a ...

  6. DNA sequences from the quagga, an extinct member of the horse family.

    Science.gov (United States)

    Higuchi, R; Bowman, B; Freiberger, M; Ryder, O A; Wilson, A C

    To determine whether DNA survives and can be recovered from the remains of extinct creatures, we have examined dried muscle from a museum specimen of the quagga, a zebra-like species (Equus quagga) that became extinct in 1883 (ref. 1). We report that DNA was extracted from this tissue in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight. Among the many clones obtained from the quagga DNA, two containing pieces of mitochondrial DNA (mtDNA) were sequenced. These sequences, comprising 229 nucleotide pairs, differ by 12 base substitutions from the corresponding sequences of mtDNA from a mountain zebra, an extant member of the genus Equus. The number, nature and locations of the substitutions imply that there has been little or no postmortem modification of the quagga DNA sequences, and that the two species had a common ancestor 3-4 Myr ago, consistent with fossil evidence concerning the age of the genus Equus.

  7. Studies of base pair sequence effects on DNA solvation based on all ...

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... [Dixit SB, Mezei M and Beveridge DL 2012 Studies of base pair sequence effects on DNA salvation based on all-atom molecular dynamics simulations. J. Biosci. 37 399–421] DOI 10.1007/s12038-012-9223-5. 1. Introduction. Solvation plays an integral role in stabilizing the structure of the DNA molecule in ...

  8. Nucleotide sequence determination of the region in adenovirus 5 DNA involved in cell transformation

    International Nuclear Information System (INIS)

    Maat, J.

    1978-01-01

    A description is given of investigations into the primary structure of the transforming region of adenovirus type 5 DNA. The phenomenon of cell transformation is discussed in general terms and the principles of a number of fairly recent techniques, which have been in use for DNA sequence determination since 1975 are dealt with. A few of the author's own techniques are described which deal both with nucleotide sequence analysis and with the determination of DNA cleavage sites of restriction endonucleases. The results are given of the mapping of cleavage sites in the HpaI-E fragment of adenovirus DNA of HpaII, HaeIII, AluI, HinfI and TaqI and of the determination of the nucleotide sequence in the transforming region of adenovirus type 5 DNA. The results of the sequence determination of the Ad5 HindIII-G fragment are discussed in relation with the investigation on the transforming proteins isolated from in vitro and in vivo synthesizing systems. Labelling procedures of DNA are described including the exonuclease III/DNA polymerase 1 method and TA polynucleotide kinase labelling of DNA fragments. (Auth.)

  9. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  10. Applications of inter simple sequence repeat (ISSR) rDNA in ...

    African Journals Online (AJOL)

    bika

    2015-04-22

    Apr 22, 2015 ... infection with Fasciola. They also found that Lymnaea snails were correlated with some factors such as prevalence of other snails and aquatic plants. Molecular techniques such as random amplified polymorphic DNA- polymerase chain reaction (RAPD-. PCR) analysis and DNA sequencing (Puslednik et ...

  11. DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing

    NARCIS (Netherlands)

    Stoddart, David; Franceschini, Lorenzo; Heron, Andrew; Bayley, Hagan; Maglia, Giovanni

    2015-01-01

    In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we

  12. Method for performing site-specific affinity fractionation for use in DNA sequencing

    Science.gov (United States)

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    1999-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  13. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob

    2016-01-01

    be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject...... from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects....

  14. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

    Directory of Open Access Journals (Sweden)

    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  15. Application of high-throughput DNA sequencing in phytopathology.

    Science.gov (United States)

    Studholme, David J; Glover, Rachel H; Boonham, Neil

    2011-01-01

    The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Copyright © 2011 by Annual Reviews. All rights reserved.

  16. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations

    NARCIS (Netherlands)

    Wagler, Patrick; Minero, Gabriel Antonio S.; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S.

    2015-01-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for

  17. GENETIC POLYMORPHISM IN GYMNODINIUM GALATHEANUM CHLOROPLAST DNA SEQUENCES AND DEVELOPMENT OF A MOLECULAR DETECTION ASSAY. (R827084)

    Science.gov (United States)

    Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtainedfrom several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses andcomparison of sequences indicate that the chloroplast sequences show a higher degree of se...

  18. Sequence-specific DNA alkylation by tandem Py-Im polyamide conjugates.

    Science.gov (United States)

    Taylor, Rhys Dylan; Kawamoto, Yusuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2014-09-01

    Tandem N-methylpyrrole-N-methylimidazole (Py-Im) polyamides with good sequence-specific DNA-alkylating activities have been designed and synthesized. Three alkylating tandem Py-Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high-resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1, which contained a β-alanine linker, displayed the most-selective sequence-specific alkylation towards the target 10 bp DNA sequence. The tandem Py-Im polyamide conjugates displayed greater sequence-specific DNA alkylation than conventional hairpin Py-Im polyamide conjugates (4 and 5). For further research, the design of tandem Py-Im polyamide conjugates could play an important role in targeting specific gene sequences. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform.

    Science.gov (United States)

    Shokralla, Shadi; Porter, Teresita M; Gibson, Joel F; Dobosz, Rafal; Janzen, Daniel H; Hallwachs, Winnie; Golding, G Brian; Hajibabaei, Mehrdad

    2015-04-17

    Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.

  20. Numerical modeling of block structure dynamics: Application to the Vrancea region and study of earthquakes sequences in the synthetic catalogs

    International Nuclear Information System (INIS)

    Soloviev, A.A.; Vorobieva, I.A.

    1995-08-01

    A seismically active region is represented as a system of absolutely rigid blocks divided by infinitely thin plane faults. The interaction of the blocks along the fault planes and with the underlying medium is viscous-elastic. The system of blocks moves as a consequence of prescribed motion of boundary blocks and the underlying medium. When for some part of a fault plane the stress surpasses a certain strength level a stress-drop (''a failure'') occurs. It can cause a failure for other parts of fault planes. The failures are considered as earthquakes. As a result of the numerical simulation a synthetic earthquake catalogue is produced. This procedure is applied for numerical modeling of dynamics of the block structure approximating the tectonic structure of the Vrancea region. By numerical experiments the values of the model parameters were obtained which supplied the synthetic earthquake catalog with the space distribution of epicenters close to the real distribution of the earthquake epicenters in the Vrancea region. The frequency-magnitude relations (Gutenberg-Richter curves) obtained for the synthetic and real catalogs have some common features. The sequences of earthquakes arising in the model are studied for some artificial structures. It is found that ''foreshocks'', ''main shocks'', and ''aftershocks'' could be detected among earthquakes forming the sequences. The features of aftershocks, foreshocks, and catalogs of main shocks are analysed. (author). 5 refs, 12 figs, 16 tabs

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

    Science.gov (United States)

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

    1999-01-01

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

  2. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There

  3. Complete mitochondrial DNA sequence of the Qianshao spotted pig.

    Science.gov (United States)

    Xu, Dong; Chai, Yu-Lan; Jiang, Juan; He, Chang-Qing; Ma, Hai-Ming

    2015-01-01

    The complete mitochondrial genome sequence of Qianshao spotted pig was first determined in this study. The mitogenome (16,700 bp) consists of 22 tRNA genes, 2 ribosomal RNA genes, 13 protein-coding genes and 1 control region (D-loop region). The complete mitochondrial genome sequence of the Qianshao spotted pig enriches data resource for further study in genetic mechanism.

  4. Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Olivier Delelis

    Full Text Available BACKGROUND: HIV-1 integrase (IN catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

  5. DNA sequence evolution in fast evolving mitochondrial DNA nad1 exons in Geraniaceae and Plantaginaceae

    NARCIS (Netherlands)

    Bakker, F.T.; Breman, F.; Merckx, V.

    2006-01-01

    Previously, nucleotide substitution rates in mitochondrial DNA of Geraniaceae and Plantaginaceae have been shown to be exceptionally high compared with other angiosperm mtDNA lineages. It has also been shown that mtDNA introns were lost in Geraniaceae and Plantaginaceae. In this study we compile 127

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  7. Tandemly repeated sequence in 5'end of mtDNA control region of ...

    African Journals Online (AJOL)

    Extensive length variability was observed in 5' end sequence of the mitochondrial DNA control region of the Japanese Spanish mackerel (Scomberomorus niphonius). This length variability was due to the presence of varying numbers of a 56-bp tandemly repeated sequence and a 46-bp insertion/deletion (indel).

  8. Studies of base pair sequence effects on DNA solvation based on all ...

    Indian Academy of Sciences (India)

    Detailed analyses of the sequence-dependent solvation and ion atmosphere of DNA are presented based on molecular dynamics (MD) simulations on all the 136 unique tetranucleotide steps obtained by the ABC consortium using the AMBER suite of programs. Significant sequence effects on solvation and ion localization ...

  9. Studies of base pair sequence effects on DNA solvation based on all

    Indian Academy of Sciences (India)

    Detailed analyses of the sequence-dependent solvation and ion atmosphere of DNA are presented based on molecular dynamics (MD) simulations on all the 136 unique tetranucleotide steps obtained by the ABC consortium using the AMBER suite of programs. Significant sequence effects on solvation and ion localization ...

  10. Open source tools to exploit DNA sequence data from livestock species

    Science.gov (United States)

    Next-Generation Sequencing (NGS) is a recent technological development that allows researchers to rapidly determine the DNA sequence of an individual. The decrease in cost of NGS has brought the technology into the realm of practical applications in livestock genomics, where it can be used to genera...

  11. Cloning, sequencing and expression of a novel xylanase cDNA from ...

    African Journals Online (AJOL)

    A strain SH 2016, capable of producing xylanase, was isolated and identified as Aspergillus awamori, based on its physiological and biochemical characteristics as well as its ITS rDNA gene sequence analysis. A xylanase gene of 591 bp was cloned from this newly isolated A. awamori and the ORF sequence predicted a ...

  12. 2D-dynamic representation of DNA sequences as a graphical tool in bioinformatics

    Science.gov (United States)

    Bielińska-Wa̧Ż, D.; Wa̧Ż, P.

    2016-10-01

    2D-dynamic representation of DNA sequences is briefly reviewed. Some new examples of 2D-dynamic graphs which are the graphical tool of the method are shown. Using the examples of the complete genome sequences of the Zika virus it is shown that the present method can be applied for the study of the evolution of viral genomes.

  13. Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

    NARCIS (Netherlands)

    Chen, E.Z.; Chiu, R.W.; Sun, H; Akolekar, R.; Chan, K.C.; Leung, T.Y.; Jiang, P.; Zheng, Y.W.; Lun, F.M.; Chan, L.Y.; Jin, Y.; Go, A.T.; Lau, E.T; To, W.W.; Leung, W.C.; Tang, R.Y.; Au-Yeung, S.K.; Lam, H.; Kung, Y.Y.; Zhang, X.; Vugt, J.M.G. van; Minekawa, R.; Tang, M.H.; Wang, J.; Oudejans, C.B.; Lau, T.K.; Nicolaides, K.H.; Lo, Y.M.

    2011-01-01

    Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due

  14. Single molecule sequencing of free DNA from maternal plasma for noninvasive trisomy 21 detection

    NARCIS (Netherlands)

    van den Oever, Jessica M. E.; Balkassmi, Sahila; Verweij, E. Joanne; van Iterson, Maarten; van Scheltema, Phebe N. Adama; Oepkes, Dick; van Lith, Jan M. M.; Hoffer, Mariëtte J. V.; den Dunnen, Johan T.; Bakker, Egbert; Boon, Elles M. J.

    2012-01-01

    Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in

  15. True single-molecule DNA sequencing of a pleistocene horse bone

    DEFF Research Database (Denmark)

    Orlando, Ludovic Antoine Alexandre; Ginolhac, Aurélien; Raghavan, Maanasa

    2011-01-01

    -preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing...

  16. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  17. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    cell embryo and the expression was monitored continuously. The expression shown here is in developing embryo and freshly hatched fish. The intensity of green colour indicate the strong expression of EGFP in all the tissues of the embryo/fry. The expression of EGPF indicates the co-expression of catfish GH cDNA and the ...

  18. DNA fingerprinting based on simple sequence repeat (SSR ...

    African Journals Online (AJOL)

    New varieties of sugarcane are protected using morphological descriptors, which have limitations in identifying morphologically similar cultivars. Development of a reliable DNA fingerprint system for identification of new varieties would contribute greatly to the breeding of these species. Microsatellite markers are tools with ...

  19. Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia).

    Science.gov (United States)

    Sinniger, Frederic; Reimer, James D; Pawlowski, Jan

    2008-12-01

    The order Zoantharia is known for its chaotic taxonomy and difficult morphological identification. One method that potentially could help for examining such troublesome taxa is DNA barcoding, which identifies species using standard molecular markers. The mitochondrial cytochrome oxidase subunit I (COI) has been utilized to great success in groups such as birds and insects; however, its applicability in many other groups is controversial. Recently, some studies have suggested that barcoding is not applicable to anthozoans. Here, we examine the use of COI and mitochondrial 16S ribosomal DNA for zoanthid identification. Despite the absence of a clear barcoding gap, our results show that for most of 54 zoanthid samples, both markers could separate samples to the species, or species group, level, particularly when easily accessible ecological or distributional data were included. Additionally, we have used the short V5 region of mt 16S rDNA to identify eight old (13 to 50 years old) museum samples. We discuss advantages and disadvantages of COI and mt 16S rDNA as barcodes for Zoantharia, and recommend that either one or both of these markers be considered for zoanthid identification in the future.

  20. MotifMark: Finding regulatory motifs in DNA sequences.

    Science.gov (United States)

    Hassanzadeh, Hamid Reza; Kolhe, Pushkar; Isbell, Charles L; Wang, May D

    2017-07-01

    The interaction between proteins and DNA is a key driving force in a significant number of biological processes such as transcriptional regulation, repair, recombination, splicing, and DNA modification. The identification of DNA-binding sites and the specificity of target proteins in binding to these regions are two important steps in understanding the mechanisms of these biological activities. A number of high-throughput technologies have recently emerged that try to quantify the affinity between proteins and DNA motifs. Despite their success, these technologies have their own limitations and fall short in precise characterization of motifs, and as a result, require further downstream analysis to extract useful and interpretable information from a haystack of noisy and inaccurate data. Here we propose MotifMark, a new algorithm based on graph theory and machine learning, that can find binding sites on candidate probes and rank their specificity in regard to the underlying transcription factor. We developed a pipeline to analyze experimental data derived from compact universal protein binding microarrays and benchmarked it against two of the most accurate motif search methods. Our results indicate that MotifMark can be a viable alternative technique for prediction of motif from protein binding microarrays and possibly other related high-throughput techniques.

  1. Sequence Dependent Interactions Between DNA and Single-Walled Carbon Nanotubes

    Science.gov (United States)

    Roxbury, Daniel

    It is known that single-stranded DNA adopts a helical wrap around a single-walled carbon nanotube (SWCNT), forming a water-dispersible hybrid molecule. The ability to sort mixtures of SWCNTs based on chirality (electronic species) has recently been demonstrated using special short DNA sequences that recognize certain matching SWCNTs of specific chirality. This thesis investigates the intricacies of DNA-SWCNT sequence-specific interactions through both experimental and molecular simulation studies. The DNA-SWCNT binding strengths were experimentally quantified by studying the kinetics of DNA replacement by a surfactant on the surface of particular SWCNTs. Recognition ability was found to correlate strongly with measured binding strength, e.g. DNA sequence (TAT)4 was found to bind 20 times stronger to the (6,5)-SWCNT than sequence (TAT)4T. Next, using replica exchange molecular dynamics (REMD) simulations, equilibrium structures formed by (a) single-strands and (b) multiple-strands of 12-mer oligonucleotides adsorbed on various SWCNTs were explored. A number of structural motifs were discovered in which the DNA strand wraps around the SWCNT and 'stitches' to itself via hydrogen bonding. Great variability among equilibrium structures was observed and shown to be directly influenced by DNA sequence and SWCNT type. For example, the (6,5)-SWCNT DNA recognition sequence, (TAT)4, was found to wrap in a tight single-stranded right-handed helical conformation. In contrast, DNA sequence T12 forms a beta-barrel left-handed structure on the same SWCNT. These are the first theoretical indications that DNA-based SWCNT selectivity can arise on a molecular level. In a biomedical collaboration with the Mayo Clinic, pathways for DNA-SWCNT internalization into healthy human endothelial cells were explored. Through absorbance spectroscopy, TEM imaging, and confocal fluorescence microscopy, we showed that intracellular concentrations of SWCNTs far exceeded those of the incubation

  2. Complete genome sequence of the mitochondrial DNA of the river lamprey, Lethenteron japonicum.

    Science.gov (United States)

    Kawai, Yuri L; Yura, Kei; Shindo, Miyuki; Kusakabe, Rie; Hayashi, Keiko; Hata, Kenichiro; Nakabayashi, Kazuhiko; Okamura, Kohji

    2015-01-01

    Lampreys are eel-like jawless fishes evolutionarily positioned between invertebrates and vertebrates, and have been used as model organisms to explore vertebrate evolution. In this study we determined the complete genome sequence of the mitochondrial DNA of the Japanese river lamprey, Lethenteron japonicum, using next-generation sequencers. The sequence was 16,272 bp in length. The gene content and order were identical to those of the sea lamprey, Petromyzon marinus, which has been the reference among lamprey species. However, the sequence similarity was less than 90%, suggesting the need for the whole-genome sequencing of L. japonicum.

  3. Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing.

    Science.gov (United States)

    Gamba, Cristina; Hanghøj, Kristian; Gaunitz, Charleen; Alfarhan, Ahmed H; Alquraishi, Saleh A; Al-Rasheid, Khaled A S; Bradley, Daniel G; Orlando, Ludovic

    2016-03-01

    The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high-throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost-effective solution for downstream applications, including DNA sequencing on HTS platforms. © 2015 John Wiley & Sons Ltd.

  4. Spliced DNA sequences in the Paramecium germline: their properties and evolutionary potential.

    Science.gov (United States)

    Catania, Francesco; McGrath, Casey L; Doak, Thomas G; Lynch, Michael

    2013-01-01

    Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic--often coding--DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation.

  5. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

    Science.gov (United States)

    Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

    2013-09-24

    Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

  6. Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

    Science.gov (United States)

    Clarke, Wayne E.; Parkin, Isobel A.; Gajardo, Humberto A.; Gerhardt, Daniel J.; Higgins, Erin; Sidebottom, Christine; Sharpe, Andrew G.; Snowdon, Rod J.; Federico, Maria L.; Iniguez-Luy, Federico L.

    2013-01-01

    Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species. PMID:24312619

  7. Ribosomal PCR and DNA sequencing for detection and identification of bacteria

    DEFF Research Database (Denmark)

    Jensen, Kristine Helander; Dargis, Rimtas; Christensen, Jens Jørgen

    2014-01-01

    The use of broad range PCR and DNA sequencing of bacterial 16S ribosomal RNA genes for routine diagnostics of bacterial infections was evaluated. Here, the results from more than 2600 analyses during a 6-year period (2003-2009) are presented. Almost half of the samples were from joints and bones......, and the second most frequent origin of samples was from the central nervous system. Overall, 26% of all samples were positive for bacterial DNA and bacterial identification was obtained in 80% of the PCR-positive samples by subsequent DNA sequencing. Ambiguous species identification was noticed among non......-haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci...

  8. Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

    Science.gov (United States)

    Riedl, Jan; Ding, Yun; Fleming, Aaron M.; Burrows, Cynthia J.

    2015-01-01

    Damage to the genome is implicated in the progression of cancer and stress-induced diseases. DNA lesions exist in low levels, and cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied and sequenced. Critical for this method is installation of either the dNaM or d5SICS nucleotides at the lesion site after processing via the base excision repair process. These marker nucleotides constitute an unnatural base pair, allowing large quantities of marked DNA to be made by PCR amplification. Sanger sequencing confirms the potential for this method to locate lesions by marking, amplifying and sequencing a lesion in the KRAS gene. Detection using the α-hemolysin nanopore is also developed to analyse the markers in individual DNA strands with the potential to identify multiple lesions per strand. PMID:26542210

  9. Applicability of Ion Torrent Colon and Lung sequencing panel on circulating cell-free DNA

    DEFF Research Database (Denmark)

    Demuth, Christina; Tranberg Madsen, Anne; Larsen, Anne Winther

    of targeted sequencing have been optimised for clinical use on FFPE, e.g. the Ion Torrent Colon and Lung panel. The size of DNA extracted from FFPE tissue is comparable with that from cfDNA. We therefore investigated the performance of the clinically relevant Ion Torrent Colon and Lung panel on cfDNA. Methods...... a baseline for the panel. Lastly, the panel was tested on 52 patient samples. Patient plasma samples are from a previously collected cohort of EGFR wild-type non-small cell lung cancer patients (ClinicalTrial.gov: NCT02043002) All samples were sequenced using the Ion Torrent Oncomine Solid Tumor DNA kit...... (Colon and Lung panel) from Thermo Fisher. Sample preparation was performed using the Ion Torrent Chef and sequencing was performed on the Personal Genome Machine (PGM) system. Data was analyzed using the Torrent Suite software, and variants called by Ion Reporter. Results: No somatic mutations were...

  10. Sequencing strategy of mitochondrial HV1 and HV2 DNA with length heteroplasmy

    DEFF Research Database (Denmark)

    Rasmussen, Erik Michael; Sørensen, E; Eriksen, Birthe

    2002-01-01

    downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mt......We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2-4 bases...

  11. Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among papovaviridae.

    OpenAIRE

    Danos, O; Katinka, M; Yaniv, M

    1982-01-01

    The complete nucleotide sequence of human papillomavirus type 1a (7811 nucleotides) has been established. The overall organization of the viral genome is different from that of other related papovaviruses (SV40, BKV, polyoma). Firstly, genetic information seems to be coded by one strand. Secondly, no significant homology is found with SV40 or polyoma coding sequence for either DNA or deducted protein sequences. The relatedness of human and bovine papillomaviruses is revealed by a conserved co...

  12. High-Coverage Long Read DNA Sequencing with the Oxford Nanopore MinION

    OpenAIRE

    Jain, Miten

    2017-01-01

    Nanopore sequencing was conceived in 1989 by Dave Deamer (UCSC). Over two decades of development from research laboratories and, later on, Oxford Nanopore Technologies resulted in the MinION nanopore sequencer. This work describes the developments in MinION nanopore sequencing and software, and technical milestones achieved since the MinION’s release in 2014. These developments include establishing DNA reads that exceed 200 kb+ lengths and direct, simultaneous detection of nucleotide modifica...

  13. Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: Relationship with DNA binding affinity and cytotoxicity

    International Nuclear Information System (INIS)

    Capranico, G.; Kohn, K.W.; Pommier, Y.; Zunino, F.

    1990-01-01

    Topoisomerase II mediated double-strand breaks produced by anthracycline analogues were studied in SV40 DNA. The compounds included doxorubicin, daunorubicin, two doxorubicin stereoisomers (4'-epimer and β-anomer), and five chromophore-modified derivatives, with a wide range of cytotoxic activity and DNA binding affinity. Cleavage of 32 P-end-labeled DNA fragments was visualized by autoradiography of agarose and polyacrylamide gels. Structure-activity relationships indicated that alterations in the chromophore structure greatly affected drug action on topoisomerase II. In particular, removal of substituents on position 4 of the D ring resulted in more active inducers of cleavage with lower DNA binding affinity. The stereochemistry between the sugar and the chromophore was also essential for activity. All the active anthracyclines induced a single region of prominent cleavage in the entire SV40 DNA, which resulted from a cluster of sites between nucleotides 4237 and 4294. DNA cleavage intensity patterns exhibited differences among analogues and were also dependent upon drug concentration. Intensity at a given site dependent on both stimulatory and suppressive effects depending upon drug concentration and DNA sequence. A good correlation was found between cytotoxicity and intensity of topoisomerase II mediated DNA breakage

  14. DNA sequencing by two-dimensional materials: As theoretical modeling meets experiments.

    Science.gov (United States)

    Liang, Lijun; Shen, Jia-Wei; Zhang, Zhisen; Wang, Qi

    2017-03-15

    Owing to their extraordinary electrical, chemical, optical, mechanical and structural properties, two-dimensional (2D) materials (mainly including graphene, boron nitride, MoS 2 etc.) have stimulated exploding interests in sensor applications. 2D-material based nanoscale DNA sequencing is a single-molecule technique with revolutionary potential. In this paper, we review the methodology of DNA sequencing based on the measurements of ionic current, force peak, and transverse electrical currents etc. by 2D materials. The advantages and disadvantages of DNA sequencing by 2D materials are discussed. Besides the recent development of experiments, we will focus on the theoretical calculations of DNA sequencing, which have been played a critical role in the development of this field. Special emphasis will focus on the disagreements between experiments and theoretical calculations, and the explanations for the discrepancy will be highlighted. Finally, some new plausible sequencing methods from computational studies will be discussed, which may be applied in the realistic DNA sequencing experiments in future. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

    Science.gov (United States)

    Álvarez-Martos, Isabel; Ferapontova, Elena E

    2017-08-05

    A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. The chemical structure of DNA sequence signals for RNA transcription

    Science.gov (United States)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  17. Analysis of the DNA binding proteins interacting with specific upstream sequences of the S. purpuratus CyI actin gene.

    Science.gov (United States)

    Ganster, R; Paul, H; Katula, K S

    1992-12-01

    The CyI actin gene of the sea urchin, Strongylocentrotus purpuratus, is regulated temporally and spatially within the cells of the early embryo. In an effort to understand the molecular basis for the CyI actin pattern of expression, we have begun analyzing the protein-DNA interactions within regions previously shown to be of potential functional importance (Katula et al., 1987). Using DNase I footprinting, 10 protected regions were identified containing both conserved and apparently novel protein binding sites. Gel mobility shift competition assays confirmed the presence of multiple protein factors which specifically recognize CyI actin upstream sequences. Determination of a relative affinity constant value (Kr) indicated that most of the protein factors preferred their respective oligonucleotide sequences vs. a synthetic competitor DNA in a range of 10(4). The highest affinity binding was observed for proteins binding to the oligonucleotide probe containing the octamer element (Kr approximately 10(6)). Heterologous gel shift competition assays were carried out to investigate the interrelatedness of the protein factors. These studies, combined with other data, indicate there are both unique and redundant protein-DNA interactions in the region being examined. Possible alterations in CyI actin DNA binding proteins were investigated during the period of CyI transcriptional activation by gel mobility shift analysis. An increase in binding activity was observed for most of the factors, indicating that early transcriptional activity of CyI actin may involve a general increase in the amount or activity of specific transcription factors. In addition, qualitative changes, as seen by alterations in the shift patterns, were observed for some of the oligonucleotide probes.

  18. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    Science.gov (United States)

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-03-22

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

  19. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fung, N.

    1998-03-27

    Since the first demonstration of the laser in the 1960`s, lasers have found numerous applications in analytical chemistry. In this work, two different applications are described, namely, DNA sequencing with capillary gel electrophoresis and single cell analysis with mass spectrometry. Two projects are described in which high-speed DNA separations with capillary gel electrophoresis were demonstrated. In the third project, flow cytometry and mass spectrometry were coupled via a laser vaporization/ionization interface and individual mammalian cells were analyzed. First, DNA Sanger fragments were separated by capillary gel electrophoresis. A separation speed of 20 basepairs per minute was demonstrated with a mixed poly(ethylene oxide) (PEO) sieving solution. In addition, a new capillary wall treatment protocol was developed in which bare (or uncoated) capillaries can be used in DNA sequencing. Second, a temperature programming scheme was used to separate DNA Sanger fragments. Third, flow cytometry and mass spectrometry were coupled with a laser vaporization/ionization interface.

  20. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  1. Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing.

    Science.gov (United States)

    Miller, Marisa E; Liberatore, Katie L; Kianian, Shahryar F

    2017-07-28

    Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in

  2. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  3. Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

    OpenAIRE

    Goddard, J M; Caput, D; Williams, S R; Martin, D W

    1983-01-01

    We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled c...

  4. Development of a Novel Technology for Label Free DNA Sequencing

    Science.gov (United States)

    2012-05-21

    biological processes and cannot be solely explained by the tight binding approximation. In real processes, the vibrations of the lattice and the...Frequency Rabi Oscillation in a Coupled-Double- Quantum-Dot Semiconductor System The mechanism of the charge transfer inside a DNA molecule under the...molecule. To this connection, in terms of density matrix theory, we have studied the Rabi oscillation of the charge transfer in a coupled- double

  5. Highly accurate fluorogenic DNA sequencing with information theory-based error correction.

    Science.gov (United States)

    Chen, Zitian; Zhou, Wenxiong; Qiao, Shuo; Kang, Li; Duan, Haifeng; Xie, X Sunney; Huang, Yanyi

    2017-12-01

    Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

  6. Highly parallel translation of DNA sequences into small molecules.

    Directory of Open Access Journals (Sweden)

    Rebecca M Weisinger

    Full Text Available A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 10(10 to 10(15 distinct molecules for the discovery of nanomolar-affinity ligands to proteins. Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands. Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons. Creating a collection of 10(10 to 10(15 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments.

  7. The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication.

    Science.gov (United States)

    Oldenburg, Delene J; Bendich, Arnold J

    2016-05-01

    The structure of a chromosomal DNA molecule may influence the way in which it is replicated and inherited. For decades plastid DNA (ptDNA) was believed to be circular, with breakage invoked to explain linear forms found upon extraction from the cell. Recent evidence indicates that ptDNA in vivo consists of linear molecules with discrete termini, although these ends were not characterized. We report the sequences of two terminal regions, End1 and End2, for maize (Zea mays L.) ptDNA. We describe structural features of these terminal regions and similarities found in other plant ptDNAs. The terminal sequences are within inverted repeat regions (leading to four genomic isomers) and adjacent to origins of replication. Conceptually, stem-loop structures may be formed following melting of the double-stranded DNA ends. Exonuclease digestion indicates that the ends in maize are unobstructed, but tobacco (Nicotiana tabacum L.) ends may have a 5'-protein. If the terminal structure of ptDNA molecules influences the retention of ptDNA, the unprotected molecular ends in mature leaves of maize may be more susceptible to degradation in vivo than the protected ends in tobacco. The terminal sequences and cumulative GC skew profiles are nearly identical for maize, wheat (Triticum aestivum L.) and rice (Oryza sativa L.), with less similarity among other plants. The linear structure is now confirmed for maize ptDNA and inferred for other plants and suggests a virus-like recombination-dependent replication mechanism for ptDNA. Plastid transformation vectors containing the terminal sequences may increase the chances of success in generating transplastomic cereals.

  8. Targeted enrichment of genomic DNA regions for next generation sequencing

    NARCIS (Netherlands)

    Mertens, F.; El-Sharawy, A.; Sauer, S.; Van Helvoort, J.; Van der Zaag, P.J.; Franke, A.; Nilsson, M.; Lehrach. H.; Brookes, A.

    2011-01-01

    In this review we discuss the latest targeted enrichment methods, and aspects of their utilization along with second generation sequencing for complex genome analysis. In doing so we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a

  9. Short Note DNA sequences from the Little Brown Bustard Eupodotis ...

    African Journals Online (AJOL)

    Taxonomic classification of birds based exclusively on morphology and plumage traits has often been found to be inconsistent with true evolutionary history when tested with molecular phylogenies based on neutrally evolving markers. Here we present cytochrome-b gene sequences for the poorly known Little Brown ...

  10. Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

    DEFF Research Database (Denmark)

    Ferreira, Elisa N; Pires, Lilian C; Parmigiani, Raphael B

    2004-01-01

    The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification...... can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within...... the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization...

  11. Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing.

    Science.gov (United States)

    Lin, Chun-Yen; Tsai, Li-Chin; Hsieh, Hsing-Mei; Huang, Chia-Hung; Yu, Yu-Jen; Tseng, Bill; Linacre, Adrian; Lee, James Chun-I

    2017-09-01

    Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184-16193, 303-315 and 568-573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Intervening sequences in a plant gene-comparison of the partial sequence of cDNA and genomic DNA of French bean phaseolin

    Science.gov (United States)

    Sun, S. M.; Slightom, J. L.; Hall, T. C.

    1981-01-01

    A plant gene coding for the major storage protein (phaseolin, G1-globulin) of the French bean was isolated from a genomic library constructed in the phage vector Charon 24A. Comparison of the nucleotide sequence of part of the gene with that of the cloned messenger RNA (cDNA) revealed the presence of three intervening sequences, all beginning with GTand ending with AG. The 5' and 3' boundaries of intervening sequences TVS-A (88 base pairs) and IVS-B (124 base pairs) are similar to those described for animal and viral genes, but the 3' boundary of IVS-C (129 base pairs) shows some differences. A sequence of 185 amino acids deduced from the cloned DMAs represents about 40% of a phaseolin polypeptide.

  13. Nucleotide sequence database comparison for Internal Transcribed Spacer 2 genetic region DNA barcode dermatophyte routine identification.

    Science.gov (United States)

    Normand, A C; Packeu, A; Cassagne, C; Hendrickx, M; Ranque, S; Piarroux, R

    2018-02-28

    Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA ITS region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification.We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study.According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton , mainly T. rubrum complex (n=184), T. interdigitale (n=40), T. tonsurans (n=26) and T. benhamiae (n=5). Other genera included Microsporum (e.g., M canis (n=21), M. audouinii (n=10) and Nannizzia gypseum (n=3), and Epidermophyton (n=3)). Species-level identification of T. rubrum complex isolates was an issue.Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly-labeled database is consulted. As many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified. Copyright © 2018 American Society for Microbiology.

  14. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins.

    Directory of Open Access Journals (Sweden)

    Adam Ameur

    2011-03-01

    Full Text Available Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

  15. Sequencing cDNAs: An Introduction to DNA Sequence Analysis in the Undergraduate Molecular Genetics Course.

    Science.gov (United States)

    Galewsky, Samuel

    2000-01-01

    Introduces a series of molecular genetics laboratories where students pick a single colony from a Drosophila melanogester embryo cDNA library and purify the plasmid, then analyze the insert through restriction digests and gel electrophoresis. (Author/YDS)

  16. Accurate mitochondrial DNA sequencing using off-target reads provides a single test to identify pathogenic point mutations.

    Science.gov (United States)

    Griffin, Helen R; Pyle, Angela; Blakely, Emma L; Alston, Charlotte L; Duff, Jennifer; Hudson, Gavin; Horvath, Rita; Wilson, Ian J; Santibanez-Koref, Mauro; Taylor, Robert W; Chinnery, Patrick F

    2014-12-01

    Mitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations. We compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects. Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and-providing there is a minimum read depth of 20-fold-rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing. This offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets.

  17. DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification.

    Science.gov (United States)

    Templeton, Jennifer E L; Brotherton, Paul M; Llamas, Bastien; Soubrier, Julien; Haak, Wolfgang; Cooper, Alan; Austin, Jeremy J

    2013-12-02

    Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging

  18. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

    Science.gov (United States)

    Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

    2016-08-01

    Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening.

    Science.gov (United States)

    Oikawa, S; Kawanishi, S

    1999-06-25

    Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H2O2 plus Cu(II) caused predominant DNA damage at the 5' site of 5'-GGG-3' in the telomere sequence. Furthermore, H2O2 plus Cu(II) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in telomere sequences more efficiently than that in non-telomere sequences. NO plus O2- efficiently caused base alteration at the 5' site of 5'-GGG-3' in the telomere sequence. It is concluded that the site-specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.

  20. Integration of hepatitis B virus DNA in chromosome-specific satellite sequences

    International Nuclear Information System (INIS)

    Shaul, Y.; Garcia, P.D.; Schonberg, S.; Rutter, W.J.

    1986-01-01

    The authors previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. They report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence

  1. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  2. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... Rattus norvegicus are 93.1, 92.5, 92.2, 91.1, 90.6 and 90.0% respectively. The amino acid sequence encoded by RPS20 gene of the Giant Panda shared a high homology (100%) with those of H. sapiens,. Mac. fascicularis, Mus musculus, B. taurus and R. norvegicus, except for P. abelii (99.88%). Primary.

  3. Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

    International Nuclear Information System (INIS)

    Sage, E.; Moustacchi, E.

    1987-01-01

    The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. The authors took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT << TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT << TA ∼ TAT << ATA < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. The results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA

  4. Experimental and theoretical studies of sequence effects on the fluctuation and melting of short DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Peyrard, M; Cuesta-Lopez, S [Universite de Lyon, Ecole Normale Superieure de Lyon, Laboratoire de Physique, CNRS UMR 5672, 46 allee d' Italie, F-69364 Lyon Cedex 07 (France); Angelov, D [Universite de Lyon, Ecole Normale Superieure de Lyon, Laboratoire de Biologie Moleculaire de la Cellule, CNRS UMR 5239, 46 allee d' Italie, F-69364 Lyon Cedex 07 (France)], E-mail: Michel.Peyrard@ens-lyon.fr

    2009-01-21

    Understanding the melting of short DNA sequences probes DNA at the scale of the genetic code and raises questions which are very different from those posed by very long sequences, which have been extensively studied. We investigate this problem by combining experiments and theory. A new experimental method allows us to make a mapping of the opening of the guanines along the sequence as a function of temperature. The results indicate that non-local effects may be important in DNA because an AT-rich region is able to influence the opening of a base pair which is about 10 base pairs away. An earlier mesoscopic model of DNA is modified to correctly describe the timescales associated with the opening of individual base pairs well below melting, and to properly take into account the sequence. Using this model to analyze some characteristic sequences for which detailed experimental data on the melting is available (Montrichok et al 2003 Europhys. Lett. 62 452), we show that we have to introduce non-local effects of AT-rich regions to get acceptable results. This brings a second indication that the influence of these highly fluctuating regions of DNA on their neighborhood can extend to some distance.

  5. Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: A technical assessment

    Directory of Open Access Journals (Sweden)

    Rachael eDoherty

    2014-05-01

    Full Text Available Recent advances made in omics technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS and more recently whole genome bisulfite sequencing (WGBS have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed.

  6. Semi-automated library preparation for high-throughput DNA sequencing platforms.

    Science.gov (United States)

    Farias-Hesson, Eveline; Erikson, Jonathan; Atkins, Alexander; Shen, Peidong; Davis, Ronald W; Scharfe, Curt; Pourmand, Nader

    2010-01-01

    Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

  7. Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

    Science.gov (United States)

    Yin, Ai-hua; Peng, Chun-fang; Zhao, Xin; Caughey, Bennett A; Yang, Jie-xia; Liu, Jian; Huang, Wei-wei; Liu, Chang; Luo, Dong-hong; Liu, Hai-liang; Chen, Yang-yi; Wu, Jing; Hou, Rui; Zhang, Mindy; Ai, Michael; Zheng, Lianghong; Xue, Rachel Q; Mai, Ming-qin; Guo, Fang-fang; Qi, Yi-ming; Wang, Dong-mei; Krawczyk, Michal; Zhang, Daniel; Wang, Yu-nan; Huang, Quan-fei; Karin, Michael; Zhang, Kang

    2015-11-24

    Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

  8. Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening.

    Science.gov (United States)

    Oikawa, S; Tada-Oikawa, S; Kawanishi, S

    2001-04-17

    Telomere shortening is associated with cellular senescence. We investigated whether UVA, which contributes to photoaging, accelerates telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately 5 times more than that in DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Catalase did not inhibit this oxidative DNA damage, indicating no or little participation of H(2)O(2) in DNA damage. These results indicate that the photoexcited endogenous photosensitizer specifically oxidizes the central guanine of 5'-GGG-3' in telomere sequence to produce 8-oxodG probably through an electron-transfer reaction. It is concluded that the site-specific damage in telomere sequence induced by UVA irradiation may participate in the increase of telomere shortening rate.

  9. Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes.

    Directory of Open Access Journals (Sweden)

    Bekim Sadikovic

    2010-12-01

    Full Text Available Mitochondrial DNA (mtDNA deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM, progressive external ophthalmoplegia (PEO, and Kearns-Sayre syndrome (KSS, to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41. Only 15% (3/20 of the young patients (<6 years old carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17 exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier

  10. Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes.

    Science.gov (United States)

    Sadikovic, Bekim; Wang, Jing; El-Hattab, Ayman W; Landsverk, Megan; Douglas, Ganka; Brundage, Ellen K; Craigen, William J; Schmitt, Eric S; Wong, Lee-Jun C

    2010-12-20

    Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 emerged in double distilled water and kept refrigerated.

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

    Science.gov (United States)

    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.

  13. Structural basis for sequence-specific recognition of DNA by TAL effectors

    KAUST Repository

    Deng, Dong

    2012-01-05

    TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

  14. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  15. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    International Nuclear Information System (INIS)

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus

  16. Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy.

    Science.gov (United States)

    Holland, Mitchell M; McQuillan, Megan R; O'Hanlon, Katherine A

    2011-06-01

    To use parallel array pyrosequencing to deconvolute mixtures of mitochondrial DNA (mtDNA) sequence and provide high resolution analysis of mtDNA heteroplasmy. The hypervariable segment 1 (HV1) of the mtDNA control region was analyzed from 30 individuals using the 454 GS Junior instrument. Mock mixtures were used to evaluate the system's ability to deconvolute mixtures and to reliably detect heteroplasmy, including heteroplasmic differences between 5 family members of the same maternal lineage. Amplicon sequencing was performed on polymerase chain reaction (PCR) products generated with primers that included multiplex identifiers (MID) and adaptors for pyrosequencing. Data analysis was performed using NextGENe® software. The analysis of an autosomal short tandem repeat (STR) locus (D18S51) and a Y-STR locus (DYS389 I/II) was performed simultaneously with a portion of HV1 to illustrate that multiplexing can encompass different markers of forensic interest. Mixtures, including heteroplasmic variants, can be detected routinely down to a component ratio of 1:250 (20 minor variant copies with a coverage rate of 5000 sequences) and can be readily detected down to 1:1000 (0.1%) with expanded coverage. Amplicon sequences from D18S51, DYS389 I/II, and the second half of HV1 were successfully partitioned and analyzed. The ability to routinely deconvolute mtDNA mixtures down to a level of 1:250 allows for high resolution analysis of mtDNA heteroplasmy, and for differentiation of individuals from the same maternal lineage. The pyrosequencing approach results in poor resolution of homopolymeric sequences, and PCR/sequencing artifacts require a filtering mechanism similar to that for STR stutter and spectral bleed through. In addition, chimeric sequences from jumping PCR must be addressed to make the method operational.

  17. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  18. New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

    Science.gov (United States)

    1987-10-13

    Insert frag- ments from p16 cDNA clones were subcloned into the phage vector Ml3mp8 or Ml3mpl9 qnd subjected to rapid sequencing using the...2), and selected cDNA insert fragments were subcloned into M13 vectors for sequencing. The sequence of the complete genome was determined, with over

  19. Computational optimisation of targeted DNA sequencing for cancer detection

    DEFF Research Database (Denmark)

    Martinez, Pierre; McGranahan, Nicholas; Birkbak, Nicolai Juul

    2013-01-01

    Despite recent progress thanks to next-generation sequencing technologies, personalised cancer medicine is still hampered by intra-tumour heterogeneity and drug resistance. As most patients with advanced metastatic disease face poor survival, there is need to improve early diagnosis. Analysing...... detection. Dividing 4,467 samples into one discovery and two independent validation cohorts, we show that up to 76% of 10 cancer types harbour at least one mutation in a panel of only 25 genes, with high sensitivity across most tumour types. Our analyses demonstrate that targeting "hotspot" regions would...

  20. Determining physical constraints in transcriptional initiationcomplexes using DNA sequence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Shultzaberger, Ryan K.; Chiang, Derek Y.; Moses, Alan M.; Eisen,Michael B.

    2007-07-01

    Eukaryotic gene expression is often under the control ofcooperatively acting transcription factors whose binding is limited bystructural constraints. By determining these structural constraints, wecan understand the "rules" that define functional cooperativity.Conversely, by understanding the rules of binding, we can inferstructural characteristics. We have developed an information theory basedmethod for approximating the physical limitations of cooperativeinteractions by comparing sequence analysis to microarray expressiondata. When applied to the coordinated binding of the sulfur amino acidregulatory protein Met4 by Cbf1 and Met31, we were able to create acombinatorial model that can correctly identify Met4 regulatedgenes.