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Sample records for quantitative biology dna

  1. Cold Spring Harbor symposia on quantitative biology. Volume XLVII, Part 2. Structures of DNA

    Energy Technology Data Exchange (ETDEWEB)

    1983-01-01

    This is Volume 2 of the proceedings of the 1982 Cold Springs Harbor Symposium on Quantitative Biology. The volume contains papers on DNA methylation, DNA replication, gene recombination, organization of genes along DNA, molecular structure and enzymology of DNA.

  2. Cold Spring Harbor symposia on quantitative biology. Volume XLVII, Part 1. Structures of DNA

    Energy Technology Data Exchange (ETDEWEB)

    1983-01-01

    The proceedings for the 47th Annual Cold Spring Harbor Symposia on Quantitative Biology are presented. This symposium focused on the Structure of DNA. Topics presented covered research in the handedness of DNA, conformational analysis, chemically modified DNA, chemical synthesis of DNA, DNA-protein interactions, DNA within nucleosomes, DNA methylation, DNA replication, gyrases and topoisomerases, recombining and mutating DNA, transcription of DNA and its regulation, the organization of genes along DNA, repetitive DNA and pseudogenes, and origins of replication, centromeres, and teleomeres.

  3. Residual DNA analysis in biologics development: review of measurement and quantitation technologies and future directions.

    Science.gov (United States)

    Wang, Xing; Morgan, Donna M; Wang, Gan; Mozier, Ned M

    2012-02-01

    Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element-1 (LINE-1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse-transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real-time polymerase chain reaction (RT-PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT-PCR is limited by the sequences to which the primers are directed. To address this, primase-based whole genome amplification is introduced herein. This paper will review the recent

  4. Abstracts of papers presented at the LVIII Cold Spring Harbor Symposium on quantitative Biology: DNA and chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    1993-12-31

    This volume contains the abstracts of oral and poster presentations made at the LVIII Cold Spring Harbor Symposium on Quantitative Biology entitles DNA & Chromosomes. The meeting was held June 2--June 9, 1993 at Cold Spring Harbor, New York.

  5. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    Energy Technology Data Exchange (ETDEWEB)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro.

  6. Programmable Quantitative DNA Nanothermometers.

    Science.gov (United States)

    Gareau, David; Desrosiers, Arnaud; Vallée-Bélisle, Alexis

    2016-07-13

    Developing molecules, switches, probes or nanomaterials that are able to respond to specific temperature changes should prove of utility for several applications in nanotechnology. Here, we describe bioinspired strategies to design DNA thermoswitches with programmable linear response ranges that can provide either a precise ultrasensitive response over a desired, small temperature interval (±0.05 °C) or an extended linear response over a wide temperature range (e.g., from 25 to 90 °C). Using structural modifications or inexpensive DNA stabilizers, we show that we can tune the transition midpoints of DNA thermometers from 30 to 85 °C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large quantitative fluorescence gains within small temperature variation (e.g., > 700% over 10 °C). Using a combination of thermoswitches of different stabilities or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly up to 50 °C in temperature range. Here, we demonstrate the reversibility, robustness, and efficiency of these programmable DNA thermometers by monitoring temperature change inside individual wells during polymerase chain reactions. We discuss the potential applications of these programmable DNA thermoswitches in various nanotechnology fields including cell imaging, nanofluidics, nanomedecine, nanoelectronics, nanomaterial, and synthetic biology.

  7. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  8. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  9. [The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

    Science.gov (United States)

    Sil'veĭstrova, O Iu; Domonova, É A; Shipulina, O Iu

    2014-04-01

    The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

  10. Quantitative approaches in developmental biology.

    Science.gov (United States)

    Oates, Andrew C; Gorfinkiel, Nicole; González-Gaitán, Marcos; Heisenberg, Carl-Philipp

    2009-08-01

    The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

  11. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    Science.gov (United States)

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Interfacing DNA nanodevices with biology

    DEFF Research Database (Denmark)

    Vinther, Mathias; Kjems, Jørgen

    2016-01-01

    in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular...... environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology....

  13. Applications of microfluidics in quantitative biology.

    Science.gov (United States)

    Bai, Yang; Gao, Meng; Wen, Lingling; He, Caiyun; Chen, Yuan; Liu, Chenli; Fu, Xiongfei; Huang, Shuqiang

    2017-10-04

    Quantitative biology is dedicated to taking advantage of quantitative reasoning and advanced engineering technologies to make biology more predictable. Microfluidics, as an emerging technique, provides new approaches to precisely control fluidic conditions on small scales and collect data in high-throughput and quantitative manners. In this review, we present the relevant applications of microfluidics to quantitative biology based on two major categories (channel-based microfluidics and droplet-based microfluidics), and their typical features. We also envision some other microfluidic techniques that may not be employed in quantitative biology right now, but have great potential in the near future. This article is protected by copyright. All rights reserved.

  14. Quantitative DNA Methylation Profiling in Cancer.

    Science.gov (United States)

    Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

    2016-01-01

    Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

  15. Quantitative assessment of DNA condensation.

    Science.gov (United States)

    Trubetskoy, V S; Slattum, P M; Hagstrom, J E; Wolff, J A; Budker, V G

    1999-02-15

    A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.

  16. Quantitative DNA Analyses for Airborne Birch Pollen.

    Directory of Open Access Journals (Sweden)

    Isabell Müller-Germann

    Full Text Available Birch trees produce large amounts of highly allergenic pollen grains that are distributed by wind and impact human health by causing seasonal hay fever, pollen-related asthma, and other allergic diseases. Traditionally, pollen forecasts are based on conventional microscopic counting techniques that are labor-intensive and limited in the reliable identification of species. Molecular biological techniques provide an alternative approach that is less labor-intensive and enables identification of any species by its genetic fingerprint. A particularly promising method is quantitative Real-Time polymerase chain reaction (qPCR, which can be used to determine the number of DNA copies and thus pollen grains in air filter samples. During the birch pollination season in 2010 in Mainz, Germany, we collected air filter samples of fine (<3 μm and coarse air particulate matter. These were analyzed by qPCR using two different primer pairs: one for a single-copy gene (BP8 and the other for a multi-copy gene (ITS. The BP8 gene was better suitable for reliable qPCR results, and the qPCR results obtained for coarse particulate matter were well correlated with the birch pollen forecasting results of the regional air quality model COSMO-ART. As expected due to the size of birch pollen grains (~23 μm, the concentration of DNA in fine particulate matter was lower than in the coarse particle fraction. For the ITS region the factor was 64, while for the single-copy gene BP8 only 51. The possible presence of so-called sub-pollen particles in the fine particle fraction is, however, interesting even in low concentrations. These particles are known to be highly allergenic, reach deep into airways and cause often severe health problems. In conclusion, the results of this exploratory study open up the possibility of predicting and quantifying the pollen concentration in the atmosphere more precisely in the future.

  17. Quantitative DNA Analyses for Airborne Birch Pollen.

    Science.gov (United States)

    Müller-Germann, Isabell; Vogel, Bernhard; Vogel, Heike; Pauling, Andreas; Fröhlich-Nowoisky, Janine; Pöschl, Ulrich; Després, Viviane R

    2015-01-01

    Birch trees produce large amounts of highly allergenic pollen grains that are distributed by wind and impact human health by causing seasonal hay fever, pollen-related asthma, and other allergic diseases. Traditionally, pollen forecasts are based on conventional microscopic counting techniques that are labor-intensive and limited in the reliable identification of species. Molecular biological techniques provide an alternative approach that is less labor-intensive and enables identification of any species by its genetic fingerprint. A particularly promising method is quantitative Real-Time polymerase chain reaction (qPCR), which can be used to determine the number of DNA copies and thus pollen grains in air filter samples. During the birch pollination season in 2010 in Mainz, Germany, we collected air filter samples of fine (<3 μm) and coarse air particulate matter. These were analyzed by qPCR using two different primer pairs: one for a single-copy gene (BP8) and the other for a multi-copy gene (ITS). The BP8 gene was better suitable for reliable qPCR results, and the qPCR results obtained for coarse particulate matter were well correlated with the birch pollen forecasting results of the regional air quality model COSMO-ART. As expected due to the size of birch pollen grains (~23 μm), the concentration of DNA in fine particulate matter was lower than in the coarse particle fraction. For the ITS region the factor was 64, while for the single-copy gene BP8 only 51. The possible presence of so-called sub-pollen particles in the fine particle fraction is, however, interesting even in low concentrations. These particles are known to be highly allergenic, reach deep into airways and cause often severe health problems. In conclusion, the results of this exploratory study open up the possibility of predicting and quantifying the pollen concentration in the atmosphere more precisely in the future.

  18. Quantitative biology: where modern biology meets physical sciences.

    Science.gov (United States)

    Shekhar, Shashank; Zhu, Lian; Mazutis, Linas; Sgro, Allyson E; Fai, Thomas G; Podolski, Marija

    2014-11-05

    Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines. © 2014 Shekhar, Zhu, Mazutis, Sgro, Fai, and Podolski. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Cold Spring Harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Volume 55 of the Cold Spring Harbor Symposium on Quantitative Biology is dedicated to the study of the brain. The symposium was subdivided into four major sections. Papers were presented in Molecular Mechanisms for Signalling; Neural Development; Sensory and Motor Systems; and Cognitive Neuroscience. Individual papers from the symposium are abstracted separately. (MHB)

  20. Cold Spring Harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1989-01-01

    This volume contains the first part of the proceeding of the 53rd Cold Springs Harbor Symposium on Quantitative Biology. This years topic was Immune Recognition. Part 1, this volume, contains papers prepared by presenters of the sessions entitled Introduction, Lymphocyte Development and Receptor Selection, and Recognition by Antibodies, Antigen Recognition by T cells. (DT)

  1. Cold Spring Harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1989-01-01

    This volume contains the second part of the proceedings of the 53rd Cold Springs Harbor Symposium on Quantitative Biology. This years topic was Immune Recognition. This volume, part 2, contains papers prepared by presenters for two sessions entitled Signals for Lymphocyte Activation, Proliferation, and Adhesion, and entitled Tolerance and Self Recognition. (DT)

  2. DNA Barcoding Investigations Bring Biology to Life

    Science.gov (United States)

    Musante, Susan

    2010-01-01

    This article describes how DNA barcoding investigations bring biology to life. Biologists recognize the power of DNA barcoding not just to teach biology through connections to the real world but also to immerse students in the exciting process of science. As an investigator in the Program for the Human Environment at Rockefeller University in New…

  3. DNA Barcoding Investigations Bring Biology to Life

    Science.gov (United States)

    Musante, Susan

    2010-01-01

    This article describes how DNA barcoding investigations bring biology to life. Biologists recognize the power of DNA barcoding not just to teach biology through connections to the real world but also to immerse students in the exciting process of science. As an investigator in the Program for the Human Environment at Rockefeller University in New…

  4. Cold spring harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1988-01-01

    For many decades, it has been clear that cells have a multitude of ways of sensing their environment and converting a plethora of external signals into measured intracellular responses. Now we realize that many first messengers do not act directly through second messengers, but instead work at the genetic level by binding to cytoplasmically located receptors, which can then bind to DNA and turn on or off the functioning of specific genes. Today, we refer to the way that external signals are passed through various cellular components as signal transduction processes, with receptors and their associated molecules known as biological transducers. Because most transducer molecules are present in very limited amounts, their study at the biochemical level until recently was at best difficult, and hypothesis as to how they functioned were almost impossible to test rigorously. Today, recombinant DNA techniques have dramatically changed the picture. Even very rare receptors are now open to analyses if their respective genes can be cloned, and virtually every month, the amino acid sequence of a new key biological transducer is established. The time was thus appropriate last June to hold a Cold Spring Harbor Symposium on the Molecular Biology of Signal Transduction. The final program consisted of 119 speakers, who spoke before an audience of 439, the largest ever yet to attend a Cold spring Harbor Symposium. This volume contains 61 papers. Individual papers are indexed separately on the energy data base.

  5. Cold spring harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1988-01-01

    For many decades, it has been clear that cells have a multitude of ways of sensing their environment and converting a plethora of external signals into measured intracellular responses. Now we realize that many first messengers do not act directly through second messengers, but instead work at the genetic level by binding to cytoplasmically located receptors, which can then bind to DNA and turn on or off the functioning of specific genes. Today, we refer to the way that external signals are passed through various cellular components as signal transduction processes, with receptors and their associated molecules known as biological transducers. Because most transducer molecules are present in very limited amounts, their study at the biochemical level until recently was at best difficult, and hypotheses as to how they functioned were almost impossible to test rigorously. Today, recombinant DNA techniques have dramatically changed the picture. Even very rare receptors are now open to analysis if their respective genes can be cloned, and virtually every month the amino acid sequence of a new key biological transducer is established. The time was thus appropriate last June to hold a Cold Spring Harbor Symposium on the Molecular Biology of Signal Transduction. The final program consisted of 119 speakers, who spoke before an audience of 439, the largest ever yet to attend a Cold Spring Harbor Symposium. This volume contains 54 papers. Individual papers are indexed separately on the energy data base.

  6. DNA nanovehicles and the biological barriers

    DEFF Research Database (Denmark)

    Okholm, Anders Hauge; Kjems, Jørgen

    2016-01-01

    DNA is emerging as a smart material to construct nanovehicles for targeted drug delivery. The programmability of Watson-Crick base paring enables construction of defined and dynamic DNA nanostructures of almost arbitrary shape and DNA can readily be functionalized with a variety of molecular...... be overcome. Here, we highlight recent strategies for DNA nanostructures in drug delivery, DNA nanovehicles, to facilitate targeting and crossing of the biological barriers. In light of this, we discuss future solutions and challenges for DNA nanovehicles to unravel their great potential to facilitate...... targeted drug delivery....

  7. Unraveling pancreatic islet biology by quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  8. Quantitative cell biology: the essential role of theory.

    Science.gov (United States)

    Howard, Jonathon

    2014-11-05

    Quantitative biology is a hot area, as evidenced by the recent establishment of institutes, graduate programs, and conferences with that name. But what is quantitative biology? What should it be? And how can it contribute to solving the big questions in biology? The past decade has seen very rapid development of quantitative experimental techniques, especially at the single-molecule and single-cell levels. In this essay, I argue that quantitative biology is much more than just the quantitation of these experimental results. Instead, it should be the application of the scientific method by which measurement is directed toward testing theories. In this view, quantitative biology is the recognition that theory and models play critical roles in biology, as they do in physics and engineering. By tying together experiment and theory, quantitative biology promises a deeper understanding of underlying mechanisms, when the theory works, or to new discoveries, when it does not.

  9. Visualizing biological reaction intermediates with DNA curtains

    Science.gov (United States)

    Zhao, Yiling; Jiang, Yanzhou; Qi, Zhi

    2017-04-01

    Single-molecule approaches have tremendous potential analyzing dynamic biological reaction with heterogeneity that cannot be effectively accessed via traditional ensemble-level biochemical approaches. The approach of deoxyribonucleic acid (DNA) curtains developed by Dr Eric Greene and his research team at Columbia University is a high-throughput single-molecule technique that utilizes fluorescent imaging to visualize protein–DNA interactions directly and allows the acquisition of statistically relevant information from hundreds or even thousands of individual reactions. This review aims to summarize the past, present, and future of DNA curtains, with an emphasis on its applications to solve important biological questions.

  10. 1, 2, 3, 4: Infusing Quantitative Literacy into Introductory Biology

    Science.gov (United States)

    Bray Speth, Elena; Momsen, Jennifer L.; Moyerbrailean, Gregory A.; Ebert-May, Diane; Long, Tammy M.; Wyse, Sara; Linton, Debra

    2010-01-01

    Biology of the twenty-first century is an increasingly quantitative science. Undergraduate biology education therefore needs to provide opportunities for students to develop fluency in the tools and language of quantitative disciplines. Quantitative literacy (QL) is important for future scientists as well as for citizens, who need to interpret…

  11. 1, 2, 3, 4: infusing quantitative literacy into introductory biology.

    Science.gov (United States)

    Speth, Elena Bray; Momsen, Jennifer L; Moyerbrailean, Gregory A; Ebert-May, Diane; Long, Tammy M; Wyse, Sara; Linton, Debra

    2010-01-01

    Biology of the twenty-first century is an increasingly quantitative science. Undergraduate biology education therefore needs to provide opportunities for students to develop fluency in the tools and language of quantitative disciplines. Quantitative literacy (QL) is important for future scientists as well as for citizens, who need to interpret numeric information and data-based claims regarding nearly every aspect of daily life. To address the need for QL in biology education, we incorporated quantitative concepts throughout a semester-long introductory biology course at a large research university. Early in the course, we assessed the quantitative skills that students bring to the introductory biology classroom and found that students had difficulties in performing simple calculations, representing data graphically, and articulating data-driven arguments. In response to students' learning needs, we infused the course with quantitative concepts aligned with the existing course content and learning objectives. The effectiveness of this approach is demonstrated by significant improvement in the quality of students' graphical representations of biological data. Infusing QL in introductory biology presents challenges. Our study, however, supports the conclusion that it is feasible in the context of an existing course, consistent with the goals of college biology education, and promotes students' development of important quantitative skills.

  12. Integrating Quantitative Thinking into an Introductory Biology Course Improves Students’ Mathematical Reasoning in Biological Contexts

    OpenAIRE

    Hester, Susan; Buxner, Sanlyn; Elfring, Lisa; Nagy, Lisa

    2014-01-01

    Recent calls for improving undergraduate biology education have emphasized the importance of students learning to apply quantitative skills to biological problems. Motivated by students’ apparent inability to transfer their existing quantitative skills to biological contexts, we designed and taught an introductory molecular and cell biology course in which we integrated application of prerequisite mathematical skills with biology content and reasoning throughout all aspects of the course. In ...

  13. On the Edge of Mathematics and Biology Integration: Improving Quantitative Skills in Undergraduate Biology Education

    Science.gov (United States)

    Feser, Jason; Vasaly, Helen; Herrera, Jose

    2013-01-01

    In this paper, the authors describe how two institutions are helping their undergraduate biology students build quantitative competencies. Incorporation of quantitative skills and reasoning in biology are framed through a discussion of two cases that both concern introductory biology courses, but differ in the complexity of the mathematics and the…

  14. On the Edge of Mathematics and Biology Integration: Improving Quantitative Skills in Undergraduate Biology Education

    Science.gov (United States)

    Feser, Jason; Vasaly, Helen; Herrera, Jose

    2013-01-01

    In this paper, the authors describe how two institutions are helping their undergraduate biology students build quantitative competencies. Incorporation of quantitative skills and reasoning in biology are framed through a discussion of two cases that both concern introductory biology courses, but differ in the complexity of the mathematics and the…

  15. Quantitative stem cell biology: the threat and the glory.

    Science.gov (United States)

    Pollard, Steven M

    2016-11-15

    Major technological innovations over the past decade have transformed our ability to extract quantitative data from biological systems at an unprecedented scale and resolution. These quantitative methods and associated large datasets should lead to an exciting new phase of discovery across many areas of biology. However, there is a clear threat: will we drown in these rivers of data? On 18th July 2016, stem cell biologists gathered in Cambridge for the 5th annual Cambridge Stem Cell Symposium to discuss 'Quantitative stem cell biology: from molecules to models'. This Meeting Review provides a summary of the data presented by each speaker, with a focus on quantitative techniques and the new biological insights that are emerging. © 2016. Published by The Company of Biologists Ltd.

  16. Is Quantitative HBsAg Measurement a Reliable Substitute for HBV DNA Quantitation?

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Mahdavi

    2015-07-01

    Conclusion: There are many factors affecting the correlation between serum HBV DNA copy number and HBsAg level such as genotype of HBV virus, phase of infection, methods of measurement, HBeAg status, and drug and types of treatment procedures. Therefore, these factors should be considered in further studies dealing with the correlation between quantitative HBV DNA and HBsAg tests.

  17. Quantum integrable systems. Quantitative methods in biology

    CERN Document Server

    Feverati, Giovanni

    2011-01-01

    Quantum integrable systems have very strong mathematical properties that allow an exact description of their energetic spectrum. From the Bethe equations, I formulate the Baxter "T-Q" relation, that is the starting point of two complementary approaches based on nonlinear integral equations. The first one is known as thermodynamic Bethe ansatz, the second one as Kl\\"umper-Batchelor-Pearce-Destri- de Vega. I show the steps toward the derivation of the equations for some of the models concerned. I study the infrared and ultraviolet limits and discuss the numerical approach. Higher rank integrals of motion can be obtained, so gaining some control on the eigenvectors. After, I discuss the Hubbard model in relation to the N = 4 supersymmetric gauge theory. The Hubbard model describes hopping electrons on a lattice. In the second part, I present an evolutionary model based on Turing machines. The goal is to describe aspects of the real biological evolution, or Darwinism, by letting evolve populations of algorithms. ...

  18. Linkage of DNA Methylation Quantitative Trait Loci to Human Cancer Risk

    Directory of Open Access Journals (Sweden)

    Holger Heyn

    2014-04-01

    Full Text Available Epigenetic regulation and, in particular, DNA methylation have been linked to the underlying genetic sequence. DNA methylation quantitative trait loci (meQTL have been identified through significant associations between the genetic and epigenetic codes in physiological and pathological contexts. We propose that interrogating the interplay between polymorphic alleles and DNA methylation is a powerful method for improving our interpretation of risk alleles identified in genome-wide association studies that otherwise lack mechanistic explanation. We integrated patient cancer risk genotype data and genome-scale DNA methylation profiles of 3,649 primary human tumors, representing 13 solid cancer types. We provide a comprehensive meQTL catalog containing DNA methylation associations for 21% of interrogated cancer risk polymorphisms. Differentially methylated loci harbor previously reported and as-yet-unidentified cancer genes. We suggest that such regulation at the DNA level can provide a considerable amount of new information about the biology of cancer-risk alleles.

  19. 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.

  20. Quantitative DNA Analysis Using Droplet Digital PCR.

    Science.gov (United States)

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  1. Identifying contributors of DNA mixtures by means of quantitative information of STR typing

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    identified using polymorphic genetic markers. However, modern typing techniques supply additional quantitative data, which contain very important information about the observed evidence. This is particularly true for cases of DNA mixtures, where more than one individual has contributed to the observed...... biological stain. This article presents a method for including the quantitative information of short tandem repeat (STR) DNA mixtures in the LR. Also, an efficient algorithmic method for finding the best matching combination of DNA mixture profiles is derived and implemented in an on-line tool for two......- and three-person DNA mixtures. Finally, we demonstrate for two-person mixtures how this best matching pair of profiles can be used in estimating the likelihood ratio using importance sampling. The reason for using importance sampling for estimating the likelihood ratio is the often vast number...

  2. Quantitative Genetic Interactions Reveal Layers of Biological Modularity

    Science.gov (United States)

    Beltrao, Pedro; Cagney, Gerard; Krogan, Nevan J.

    2010-01-01

    In the past, biomedical research has embraced a reductionist approach, primarily focused on characterizing the individual components that comprise a system of interest. Recent technical developments have significantly increased the size and scope of data describing biological systems. At the same time, advances in the field of systems biology have evoked a broader view of how the underlying components are interconnected. In this essay, we discuss how quantitative genetic interaction mapping has enhanced our view of biological systems, allowing a deeper functional interrogation at different biological scales. PMID:20510918

  3. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  4. Integrating quantitative thinking into an introductory biology course improves students' mathematical reasoning in biological contexts.

    Science.gov (United States)

    Hester, Susan; Buxner, Sanlyn; Elfring, Lisa; Nagy, Lisa

    2014-01-01

    Recent calls for improving undergraduate biology education have emphasized the importance of students learning to apply quantitative skills to biological problems. Motivated by students' apparent inability to transfer their existing quantitative skills to biological contexts, we designed and taught an introductory molecular and cell biology course in which we integrated application of prerequisite mathematical skills with biology content and reasoning throughout all aspects of the course. In this paper, we describe the principles of our course design and present illustrative examples of course materials integrating mathematics and biology. We also designed an outcome assessment made up of items testing students' understanding of biology concepts and their ability to apply mathematical skills in biological contexts and administered it as a pre/postcourse test to students in the experimental section and other sections of the same course. Precourse results confirmed students' inability to spontaneously transfer their prerequisite mathematics skills to biological problems. Pre/postcourse outcome assessment comparisons showed that, compared with students in other sections, students in the experimental section made greater gains on integrated math/biology items. They also made comparable gains on biology items, indicating that integrating quantitative skills into an introductory biology course does not have a deleterious effect on students' biology learning.

  5. Cold Spring Harbor symposia on quantitative biology: Volume 51, Molecular biology of Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    1986-01-01

    Thirteen years marked the time between the discovery of the double helix in 1953 and the elucidation of the genetic code in 1966. A similar interval has now passed since the development by Cohen and Boyer of a simple procedure for the cloning of selective DNA fragments. The scientific advances made possible by the subsequent modification and elaboration of these original cloning procedures now amaze, stimulate, and increasingly often overwhelm us. Facts that until recently were virtually unobtainable now flow forth almost effortlessly. Most excitingly, the frenetic pace of these new discoveries, instead of marking the impending end of a glorious moment of learning, give every indication of opening up scientific frontiers that will take hundreds if not thousands of years to explore thoroughly. This new era of enlightenment is nowhere more apparent than in our newfound ability to study ourselves at the molecular level. This volume is the first of two collections of papers submitted by the contributors to the Cold Spring Harbor symposia on quantitative biology for 1986 - molecular biology of Homo sapiens. Contained in this collection are 80 papers grouped into sessions entitled Human Gene Map, Genetic Diagnosis, Human Evolution, and Drugs Made Off Human Genes.

  6. Universal platform for quantitative analysis of DNA transposition

    Directory of Open Access Journals (Sweden)

    Pajunen Maria I

    2010-11-01

    Full Text Available Abstract Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. Results Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. Conclusions The established universal papillation assay platform should be widely applicable to a

  7. Reproducible quantitative proteotype data matrices for systems biology.

    Science.gov (United States)

    Röst, Hannes L; Malmström, Lars; Aebersold, Ruedi

    2015-11-05

    Historically, many mass spectrometry-based proteomic studies have aimed at compiling an inventory of protein compounds present in a biological sample, with the long-term objective of creating a proteome map of a species. However, to answer fundamental questions about the behavior of biological systems at the protein level, accurate and unbiased quantitative data are required in addition to a list of all protein components. Fueled by advances in mass spectrometry, the proteomics field has thus recently shifted focus toward the reproducible quantification of proteins across a large number of biological samples. This provides the foundation to move away from pure enumeration of identified proteins toward quantitative matrices of many proteins measured across multiple samples. It is argued here that data matrices consisting of highly reproducible, quantitative, and unbiased proteomic measurements across a high number of conditions, referred to here as quantitative proteotype maps, will become the fundamental currency in the field and provide the starting point for downstream biological analysis. Such proteotype data matrices, for example, are generated by the measurement of large patient cohorts, time series, or multiple experimental perturbations. They are expected to have a large effect on systems biology and personalized medicine approaches that investigate the dynamic behavior of biological systems across multiple perturbations, time points, and individuals. © 2015 Röst et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Infusing Quantitative Approaches throughout the Biological Sciences Curriculum

    Science.gov (United States)

    Thompson, Katerina V.; Cooke, Todd J.; Fagan, William F.; Gulick, Denny; Levy, Doron; Nelson, Kären C.; Redish, Edward F.; Smith, Robert F.; Presson, Joelle

    2013-01-01

    A major curriculum redesign effort at the University of Maryland is infusing all levels of our undergraduate biological sciences curriculum with increased emphasis on interdisciplinary connections and quantitative approaches. The curriculum development efforts have largely been guided by recommendations in the National Research Council's "Bio…

  9. Infusing Quantitative Approaches throughout the Biological Sciences Curriculum

    Science.gov (United States)

    Thompson, Katerina V.; Cooke, Todd J.; Fagan, William F.; Gulick, Denny; Levy, Doron; Nelson, Kären C.; Redish, Edward F.; Smith, Robert F.; Presson, Joelle

    2013-01-01

    A major curriculum redesign effort at the University of Maryland is infusing all levels of our undergraduate biological sciences curriculum with increased emphasis on interdisciplinary connections and quantitative approaches. The curriculum development efforts have largely been guided by recommendations in the National Research Council's…

  10. High-Content Screening for Quantitative Cell Biology.

    Science.gov (United States)

    Mattiazzi Usaj, Mojca; Styles, Erin B; Verster, Adrian J; Friesen, Helena; Boone, Charles; Andrews, Brenda J

    2016-08-01

    High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future.

  11. Logic Gate Operation by DNA Translocation through Biological Nanopores.

    Directory of Open Access Journals (Sweden)

    Hiroki Yasuga

    Full Text Available Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs "1" and "0" as single-stranded DNA (ssDNA that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.

  12. Cold Spring Harbor symposia on quantitative biology: Volume 51, Molecular biology of /ital Homo sapiens/

    Energy Technology Data Exchange (ETDEWEB)

    1986-01-01

    This volume is the second part of a collection of papers submitted by the participants to the 1986 Cold Spring Harbor Symposium on Quantitative Biology entitled Molecular Biology of /ital Homo sapiens/. The 49 papers included in this volume are grouped by subject into receptors, human cancer genes, and gene therapy. (DT)

  13. Protocol for quantitative tracing of surface water with synthetic DNA

    Science.gov (United States)

    Foppen, J. W.; Bogaard, T. A.

    2012-04-01

    , the field tests were performed with salt and deuterium as tracer. To study possible decay by sunlight and/or microbial activity for synthetic DNA, immediately in the field and for the duration of the entire experiment, we carried out batch experiments. All samples were stored in a 1.5 ml Eppendorf vial in a cool-box in dry ice (-80°C). Quantitative PCR on a Mini Opticon (Bio Rad, Hercules, CA, USA) was carried out to determine DNA concentrations in the samples. Results showed the importance of a strict protocol for working with ssDNA as a tracer for quantitative tracing, since ssDNA interacts with surface areas of glass and plastic, depending on water quality and ionic strength. Interaction with the sediment and decay due to sunlight and/or microbial activity was negligible in most cases. The ssDNA protocol was then tested in natural streams. Promising results were obtained using ssDNA as quantitative tracer. The breakthrough curves using ssDNA were similar to the ones of salt or deuterium. We will present the revised protocol to use ssDNA for multi-tracing experiments in natural streams and discuss the opportunities and limitations.

  14. Quantitation of DNA methylation by melt curve analysis

    Directory of Open Access Journals (Sweden)

    Jones Michael E

    2009-04-01

    Full Text Available Abstract Background Methylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler. Methods We first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample. Results For validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues. Conclusion We have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The

  15. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    Directory of Open Access Journals (Sweden)

    Erin M Siegel

    Full Text Available Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2. A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003. Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  16. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    Science.gov (United States)

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  17. Lessons Learned from Quantitative Dynamical Modeling in Systems Biology

    Science.gov (United States)

    Bachmann, Julie; Matteson, Andrew; Schelke, Max; Kaschek, Daniel; Hug, Sabine; Kreutz, Clemens; Harms, Brian D.; Theis, Fabian J.; Klingmüller, Ursula; Timmer, Jens

    2013-01-01

    Due to the high complexity of biological data it is difficult to disentangle cellular processes relying only on intuitive interpretation of measurements. A Systems Biology approach that combines quantitative experimental data with dynamic mathematical modeling promises to yield deeper insights into these processes. Nevertheless, with growing complexity and increasing amount of quantitative experimental data, building realistic and reliable mathematical models can become a challenging task: the quality of experimental data has to be assessed objectively, unknown model parameters need to be estimated from the experimental data, and numerical calculations need to be precise and efficient. Here, we discuss, compare and characterize the performance of computational methods throughout the process of quantitative dynamic modeling using two previously established examples, for which quantitative, dose- and time-resolved experimental data are available. In particular, we present an approach that allows to determine the quality of experimental data in an efficient, objective and automated manner. Using this approach data generated by different measurement techniques and even in single replicates can be reliably used for mathematical modeling. For the estimation of unknown model parameters, the performance of different optimization algorithms was compared systematically. Our results show that deterministic derivative-based optimization employing the sensitivity equations in combination with a multi-start strategy based on latin hypercube sampling outperforms the other methods by orders of magnitude in accuracy and speed. Finally, we investigated transformations that yield a more efficient parameterization of the model and therefore lead to a further enhancement in optimization performance. We provide a freely available open source software package that implements the algorithms and examples compared here. PMID:24098642

  18. Lessons learned from quantitative dynamical modeling in systems biology.

    Directory of Open Access Journals (Sweden)

    Andreas Raue

    Full Text Available Due to the high complexity of biological data it is difficult to disentangle cellular processes relying only on intuitive interpretation of measurements. A Systems Biology approach that combines quantitative experimental data with dynamic mathematical modeling promises to yield deeper insights into these processes. Nevertheless, with growing complexity and increasing amount of quantitative experimental data, building realistic and reliable mathematical models can become a challenging task: the quality of experimental data has to be assessed objectively, unknown model parameters need to be estimated from the experimental data, and numerical calculations need to be precise and efficient. Here, we discuss, compare and characterize the performance of computational methods throughout the process of quantitative dynamic modeling using two previously established examples, for which quantitative, dose- and time-resolved experimental data are available. In particular, we present an approach that allows to determine the quality of experimental data in an efficient, objective and automated manner. Using this approach data generated by different measurement techniques and even in single replicates can be reliably used for mathematical modeling. For the estimation of unknown model parameters, the performance of different optimization algorithms was compared systematically. Our results show that deterministic derivative-based optimization employing the sensitivity equations in combination with a multi-start strategy based on latin hypercube sampling outperforms the other methods by orders of magnitude in accuracy and speed. Finally, we investigated transformations that yield a more efficient parameterization of the model and therefore lead to a further enhancement in optimization performance. We provide a freely available open source software package that implements the algorithms and examples compared here.

  19. From DNA sequence to transcriptional behaviour: a quantitative approach.

    Science.gov (United States)

    Segal, Eran; Widom, Jonathan

    2009-07-01

    Complex transcriptional behaviours are encoded in the DNA sequences of gene regulatory regions. Advances in our understanding of these behaviours have been recently gained through quantitative models that describe how molecules such as transcription factors and nucleosomes interact with genomic sequences. An emerging view is that every regulatory sequence is associated with a unique binding affinity landscape for each molecule and, consequently, with a unique set of molecule-binding configurations and transcriptional outputs. We present a quantitative framework based on existing methods that unifies these ideas. This framework explains many experimental observations regarding the binding patterns of factors and nucleosomes and the dynamics of transcriptional activation. It can also be used to model more complex phenomena such as transcriptional noise and the evolution of transcriptional regulation.

  20. Extracting biological knowledge from DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    De La Vega, F.M. [CINVESTAV-IPN (Mexico); Thieffry, D. [Universite Libre de Bruxelles, Rhode-Saint-Genese (Belgium)]|[Universidad Nacional Autonoma de Mexico, Morelos (Mexico); Collado-Vides, J. [Universidad Nacional Autonoma de Mexico, Morelos (Mexico)

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  1. Quantitation of DNA Adducts Induced by 1,3-Butadiene

    Science.gov (United States)

    Sangaraju, Dewakar; Villalta, Peter W.; Wickramaratne, Susith; Swenberg, James; Tretyakova, Natalia

    2014-07-01

    Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI+-HRMS3 analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 106 nucleotides in HT1080 cells treated with 0.5-10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 106 nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI+-HRMS3 Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

  2. Model of biological quantum logic in DNA.

    Science.gov (United States)

    Mihelic, F Matthew

    2013-08-02

    The DNA molecule has properties that allow it to act as a quantum logic processor. It has been demonstrated that there is coherent conduction of electrons longitudinally along the DNA molecule through pi stacking interactions of the aromatic nucleotide bases, and it has also been demonstrated that electrons moving longitudinally along the DNA molecule are subject to a very efficient electron spin filtering effect as the helicity of the DNA molecule interacts with the spin of the electron. This means that, in DNA, electrons are coherently conducted along a very efficient spin filter. Coherent electron spin is held in a logically and thermodynamically reversible chiral symmetry between the C2-endo and C3-endo enantiomers of the deoxyribose moiety in each nucleotide, which enables each nucleotide to function as a quantum gate. The symmetry break that provides for quantum decision in the system is determined by the spin direction of an electron that has an orbital angular momentum that is sufficient to overcome the energy barrier of the double well potential separating the C2-endo and C3-endo enantiomers, and that enantiomeric energy barrier is appropriate to the Landauer limit of the energy necessary to randomize one bit of information.

  3. Model of Biological Quantum Logic in DNA

    Directory of Open Access Journals (Sweden)

    F. Matthew Mihelic

    2013-08-01

    Full Text Available The DNA molecule has properties that allow it to act as a quantum logic processor. It has been demonstrated that there is coherent conduction of electrons longitudinally along the DNA molecule through pi stacking interactions of the aromatic nucleotide bases, and it has also been demonstrated that electrons moving longitudinally along the DNA molecule are subject to a very efficient electron spin filtering effect as the helicity of the DNA molecule interacts with the spin of the electron. This means that, in DNA, electrons are coherently conducted along a very efficient spin filter. Coherent electron spin is held in a logically and thermodynamically reversible chiral symmetry between the C2-endo and C3-endo enantiomers of the deoxyribose moiety in each nucleotide, which enables each nucleotide to function as a quantum gate. The symmetry break that provides for quantum decision in the system is determined by the spin direction of an electron that has an orbital angular momentum that is sufficient to overcome the energy barrier of the double well potential separating the C2-endo and C3-endo enantiomers, and that enantiomeric energy barrier is appropriate to the Landauer limit of the energy necessary to randomize one bit of information.

  4. Quantitative mass spectrometry of unconventional human biological matrices

    Science.gov (United States)

    Dutkiewicz, Ewelina P.; Urban, Pawel L.

    2016-10-01

    The development of sensitive and versatile mass spectrometric methodology has fuelled interest in the analysis of metabolites and drugs in unconventional biological specimens. Here, we discuss the analysis of eight human matrices-hair, nail, breath, saliva, tears, meibum, nasal mucus and skin excretions (including sweat)-by mass spectrometry (MS). The use of such specimens brings a number of advantages, the most important being non-invasive sampling, the limited risk of adulteration and the ability to obtain information that complements blood and urine tests. The most often studied matrices are hair, breath and saliva. This review primarily focuses on endogenous (e.g. potential biomarkers, hormones) and exogenous (e.g. drugs, environmental contaminants) small molecules. The majority of analytical methods used chromatographic separation prior to MS; however, such a hyphenated methodology greatly limits analytical throughput. On the other hand, the mass spectrometric methods that exclude chromatographic separation are fast but suffer from matrix interferences. To enable development of quantitative assays for unconventional matrices, it is desirable to standardize the protocols for the analysis of each specimen and create appropriate certified reference materials. Overcoming these challenges will make analysis of unconventional human biological matrices more common in a clinical setting. This article is part of the themed issue 'Quantitative mass spectrometry'.

  5. Quantitative analysis of TALE-DNA interactions suggests polarity effects.

    Science.gov (United States)

    Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

    2013-04-01

    Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

  6. Cold Spring Harbor symposia on quantitative biology: Volume L, Molecular biology of development

    Energy Technology Data Exchange (ETDEWEB)

    1985-01-01

    This volume contains contributions by contributors to the 1985 Cold Springs Harbor Symposium on Quantitative Biology. This year's theme was Molecular Biology of Development. The volume consists of 104 articles organized by content into sections entitled Nuclear/Cytoplasmic Interactions in Early Development; Lineage and Segmentation/Pattern Formation; Homeotic Mutants; Homeo Boxes; Tissue Specificity/Position Effects; Expression of Genes Introduced into Transgenic Mice; Induced Developmental Defects; Control of Gene Expression; Sex Determination; Cell-cycle Effects; Pluripotent Cells/Oncogenes; Cellular Differentiation; and Developmental Neurobiology.

  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. 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...... with the development and improvement of DNA editing strategies,compatible with other DNA assembly methodologies, genome engineering and,eventually, automation processes. Expanding and optimizing the synbio toolkit has important applications in pathway optimization for metabolic engineering, design and characterization...

  9. Environmental DNA for wildlife biology and biodiversity monitoring

    DEFF Research Database (Denmark)

    Bohmann, Kristine; Evans, Alice; Gilbert, M. Thomas P.

    2014-01-01

    Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent...... tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through...

  10. Quantitative characterization of nanoparticle agglomeration within biological media

    Energy Technology Data Exchange (ETDEWEB)

    Hondow, Nicole, E-mail: n.hondow@leeds.ac.uk; Brydson, Rik [University of Leeds, Institute for Materials Research (United Kingdom); Wang, Peiyi [University of Leeds, Institute of Molecular and Cellular Biology (United Kingdom); Holton, Mark D.; Brown, M. Rowan; Rees, Paul; Summers, Huw D. [Swansea University, Centre for Nanohealth, College of Engineering (United Kingdom); Brown, Andy [University of Leeds, Institute for Materials Research (United Kingdom)

    2012-07-15

    Quantitative analysis of nanoparticle dispersion state within biological media is essential to understanding cellular uptake and the roles of diffusion, sedimentation, and endocytosis in determining nanoparticle dose. The dispersion of polymer-coated CdTe/ZnS quantum dots in water and cell growth medium with and without fetal bovine serum was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Characterization by TEM of samples prepared by plunge freezing the blotted solutions into liquid ethane was sensitive to the dispersion state of the quantum dots and enabled measurement of agglomerate size distributions even in the presence of serum proteins where DLS failed. In addition, TEM showed a reduced packing fraction of quantum dots per agglomerate when dispersed in biological media and serum compared to just water, highlighting the effect of interactions between the media, serum proteins, and the quantum dots. The identification of a heterogeneous distribution of quantum dots and quantum dot agglomerates in cell growth medium and serum by TEM will enable correlation with the previously reported optical metrology of in vitro cellular uptake of this quantum dot dispersion. In this paper, we present a comparative study of TEM and DLS and show that plunge-freeze TEM provides a robust assessment of nanoparticle agglomeration state.

  11. Reshaping Plant Biology: Qualitative and Quantitative Descriptors for Plant Morphology

    Science.gov (United States)

    Balduzzi, Mathilde; Binder, Brad M.; Bucksch, Alexander; Chang, Cynthia; Hong, Lilan; Iyer-Pascuzzi, Anjali S.; Pradal, Christophe; Sparks, Erin E.

    2017-01-01

    An emerging challenge in plant biology is to develop qualitative and quantitative measures to describe the appearance of plants through the integration of mathematics and biology. A major hurdle in developing these metrics is finding common terminology across fields. In this review, we define approaches for analyzing plant geometry, topology, and shape, and provide examples for how these terms have been and can be applied to plants. In leaf morphological quantifications both geometry and shape have been used to gain insight into leaf function and evolution. For the analysis of cell growth and expansion, we highlight the utility of geometric descriptors for understanding sepal and hypocotyl development. For branched structures, we describe how topology has been applied to quantify root system architecture to lend insight into root function. Lastly, we discuss the importance of using morphological descriptors in ecology to assess how communities interact, function, and respond within different environments. This review aims to provide a basic description of the mathematical principles underlying morphological quantifications. PMID:28217137

  12. Thermodynamic Rule Determining the Biological DNA Information Capacity

    CERN Document Server

    Widom, A; Srivastava, Y N; Sivasubramanian, S; Valenzi, V I

    2012-01-01

    A rigorous thermodynamic expression is derived for the total biological information capacity per unit length of a DNA molecule. The total information includes the usual four letter coding sequence information plus that excess information coding often erroneously referred to as "junk". We conclude that the currently understood human DNA code is about a hundred megabyte program written on a molecule with about a ten gigabyte memory. By far, most of the programing code is not presently understood.

  13. 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.

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

    Science.gov (United States)

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

    2016-02-19

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

  15. Molecular Dynamics Simulations of DNA Translocation through a biological Nanopore

    OpenAIRE

    Barder, Simen Eidsmo

    2012-01-01

    Experimental and simulation studies of nucleic acid transport through nanosized channels, both biological and synthetic, has become a rapidly growing research area over the last decade. While the utilization of the alpha-hemolysin channel as a sequencing device is soon to be realized, other biological nanochannels may hold advantages that are yet unknown. Motivated by this, the first reported molecular dynamics simulations of DNA translocation through a connexon 26 channel were accomplished, ...

  16. Charge Migration in DNA Perspectives from Physics, Chemistry, and Biology

    CERN Document Server

    Chakraborty, Tapash

    2007-01-01

    Charge migration through DNA has been the focus of considerable interest in recent years. A deeper understanding of the nature of charge transfer and transport along the double helix is important in fields as diverse as physics, chemistry and nanotechnology. It has also important implications in biology, in particular in DNA damage and repair. This book presents contributions from an international team of researchers active in this field. It contains a wide range of topics that includes the mathematical background of the quantum processes involved, the role of charge transfer in DNA radiation damage, a new approach to DNA sequencing, DNA photonics, and many others. This book should be of value to researchers in condensed matter physics, chemical physics, physical chemistry, and nanoscale sciences.

  17. A Quantitative Tool for Producing DNA-Based Diagnostic Arrays

    Energy Technology Data Exchange (ETDEWEB)

    Tom J. Whitaker

    2008-07-11

    The purpose of this project was to develop a precise, quantitative method to analyze oligodeoxynucleotides (ODNs) on an array to enable a systematic approach to quality control issues affecting DNA microarrays. Two types of ODN's were tested; ODN's formed by photolithography and ODN's printed onto microarrays. Initial work in Phase I, performed in conjunction with Affymetrix, Inc. who has a patent on a photolithographic in situ technique for creating DNA arrays, was very promising but did seem to indicate that the atomization process was not complete. Soon after Phase II work was under way, Affymetrix had further developed fluorescent methods and indicated they were no longer interested in our resonance ionization technique. This was communicated to the program manager and it was decided that the project would continue and be focused on printed ODNs. The method being tested is called SIRIS, Sputter-Initiated Resonance Ionization Spectroscopy. SIRIS has been shown to be a highly sensitive, selective, and quantitative tool for atomic species. This project was aimed at determining if an ODN could be labeled in such a way that SIRIS could be used to measure the label and thus provide quantitative measurements of the ODN on an array. One of the largest problems in this study has been developing a method that allows us to know the amount of an ODN on a surface independent of the SIRIS measurement. Even though we could accurately determine the amount of ODN deposited on a surface, the amount that actually attached to the surface is very difficult to measure (hence the need for a quantitative tool). A double-labeling procedure was developed in which 33P and Pt were both used to label ODNs. The radioactive 33P could be measured by a proportional counter that maps the counts in one dimension. This gave a good measurement of the amount of ODN remaining on a surface after immobilization and washing. A second label, Pt, was attached to guanine nucleotides in the

  18. Visual characterization and quantitative measurement of artemisinin-induced DNA breakage

    Energy Technology Data Exchange (ETDEWEB)

    Cai Huaihong [Bionanotechnology Lab, and Department of Chemistry, Jinan University, Guangzhou 510632 (China); Yang Peihui [Bionanotechnology Lab, and Department of Chemistry, Jinan University, Guangzhou 510632 (China)], E-mail: typh@jnu.edu.cn; Chen Jianan [Bionanotechnology Lab, and Department of Chemistry, Jinan University, Guangzhou 510632 (China); Liang Zhihong [Experiment and Technology Center, Jinan University, Guangzhou 510632 (China); Chen Qiongyu [Institute of Genetic Engineering, Jinan University, Guangzhou 510632 (China); Cai Jiye [Bionanotechnology Lab, and Department of Chemistry, Jinan University, Guangzhou 510632 (China)], E-mail: tjycai@jnu.edu.cn

    2009-05-01

    DNA conformational change and breakage induced by artemisinin, a traditional Chinese herbal medicine, have been visually characterized and quantitatively measured by the multiple tools of electrochemistry, UV-vis absorption spectroscopy, atomic force microscopy (AFM), and DNA electrophoresis. Electrochemical and spectroscopic results confirm that artemisinin can intercalate into DNA double helix, which causes DNA conformational changes. AFM imaging vividly demonstrates uneven DNA strand breaking induced by QHS interaction. To assess these DNA breakages, quantitative analysis of the extent of DNA breakage has been performed by analyzing AFM images. Basing on the statistical analysis, the occurrence of DNA breaks is found to depend on the concentration of artemisinin. DNA electrophoresis further validates that the intact DNA molecules are unwound due to the breakages occur at the single strands. A reliable scheme is proposed to explain the process of artemisinin-induced DNA cleavage. These results can provide further information for better understanding the anticancer activity of artemisinin.

  19. Biological Dynamics Markup Language (BDML): an open format for representing quantitative biological dynamics data.

    Science.gov (United States)

    Kyoda, Koji; Tohsato, Yukako; Ho, Kenneth H L; Onami, Shuichi

    2015-04-01

    Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  20. Quantitative DNA interstrand cross-link formation by coumarin and thymine: structure determination, sequence effect, and fluorescence detection.

    Science.gov (United States)

    Sun, Huabing; Fan, Heli; Peng, Xiaohua

    2014-12-01

    The coumarin analogues have been widely utilized in medicine, biology, biochemistry, and material sciences. Here, we report a detailed study on the reactivity of coumarins toward DNA. A series of coumarin analogues were synthesized and incorporated into oligodeoxynucleotides. A photoinduced [2 + 2] cycloaddition occurs between the coumarin moiety and the thymidine upon 350 nm irradiation forming both syn- and anti-cyclobutane adducts (17 and 18), which are photoreversible by 254/350 nm irradiation in DNA. Quantitative DNA interstrand cross-link (ICL) formation was observed with the coumarin moieties containing a flexible two-carbon or longer chain. DNA cross-linking by coumarins shows a kinetic preference when flanked by an A:T base pair as opposed to a G:C pair. An efficient photoinduced electron transfer between coumarin and dG slows down ICL formation. ICL formation quenches the fluorescence of coumarin, which, for the first time, enables fast, easy, and real-time monitoring of DNA cross-linking and photoreversibility via fluorescence spectroscopy. It can be used to detect the transversion mutation between pyrimidines and purines. Overall, this work provides new insights into the biochemical properties and possible toxicity of coumarins. A quantitative, fluorescence-detectable, and photoswitchable DNA cross-linking reaction of the coumarin moieties can potentially serve as mechanistic probes and tools for bioresearch without disrupting native biological environment.

  1. Teaching Biology around Themes: Teach Proteins and DNA Together.

    Science.gov (United States)

    Offner, Susan

    1992-01-01

    Proposes as a unifying theme for high school biology the question of "how chromosomes determine what we are." Describes a sequence of lessons in which students learn about proteins, enzymes, and amino acids. Includes three dry laboratory exercises to demonstrate the DNA sequences for sickle cell anemia and cystic fibrosis. (MDH)

  2. The DNA-damage response in human biology and disease

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management....

  3. DNA confinement in nanochannels: physics and biological applications

    Science.gov (United States)

    Reisner, Walter; Pedersen, Jonas N.; Austin, Robert H.

    2012-10-01

    DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

  4. Biological evolution of replicator systems: towards a quantitative approach.

    Science.gov (United States)

    Martin, Osmel; Horvath, J E

    2013-04-01

    The aim of this work is to study the features of a simple replicator chemical model of the relation between kinetic stability and entropy production under the action of external perturbations. We quantitatively explore the different paths leading to evolution in a toy model where two independent replicators compete for the same substrate. To do that, the same scenario described originally by Pross (J Phys Org Chem 17:312-316, 2004) is revised and new criteria to define the kinetic stability are proposed. Our results suggest that fast replicator populations are continually favored by the effects of strong stochastic environmental fluctuations capable to determine the global population, the former assumed to be the only acting evolution force. We demonstrate that the process is continually driven by strong perturbations only, and that population crashes may be useful proxies for these catastrophic environmental fluctuations. As expected, such behavior is particularly enhanced under very large scale perturbations, suggesting a likely dynamical footprint in the recovery patterns of new species after mass extinction events in the Earth's geological past. Furthermore, the hypothesis that natural selection always favors the faster processes may give theoretical support to different studies that claim the applicability of maximum principles like the Maximum Metabolic Flux (MMF) or Maximum Entropy Productions Principle (MEPP), seen as the main goal of biological evolution.

  5. Nanoliter scale microbioreactor array for quantitative cell biology.

    Science.gov (United States)

    Lee, Philip J; Hung, Paul J; Rao, Vivek M; Lee, Luke P

    2006-05-05

    A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring.

  6. Interfacing DNA nanodevices with biology: challenges, solutions and perspectives

    Science.gov (United States)

    Vinther, Mathias; Kjems, Jørgen

    2016-08-01

    The cellular machinery performs millions of complex reactions with extreme precision at nanoscale. From studying these reactions, scientists have become inspired to build artificial nanosized molecular devices with programmed functions. One of the fundamental tools in designing and creating these nanodevices is molecular self-assembly. In nature, deoxyribonucleic acid (DNA) is inarguably one of the most remarkable self-assembling molecules. Governed by the Watson-Crick base-pairing rules, DNA assembles with a structural reliability and predictability based on sequence composition unlike any other complex biological polymer. This consistency has enabled rational design of hundreds of two- and three-dimensional shapes with a molecular precision and homogeneity not preceded by any other known technology at the nanometer scale. During the last two decades, DNA nanotechnology has undergone a rapid evolution pioneered by the work of Nadrian Seeman (Kallenbach et al 1983 Nature 205 829-31). Especially the introduction of the versatile DNA Origami technique by Rothemund (2006 Nature 440 297-302) led to an efflorescence of new DNA-based self-assembled nanostructures (Andersen et al 2009 Nature 459 73-6, Douglas et al 2009 Nature 459 414-8, Dietz et al 2009 Science 325 725-30, Han et al 2011 Science 332 342-6, Iinuma et al 2014 Science 344 65-9), and variations of this technique have contributed to an increasing repertoire of DNA nanostructures (Wei et al 2012 Nature 485 623-6, Ke et al 2012 Science 338 1177-83, Benson et al 2015 Nature 523 441-4, Zhang et al 2015 Nat. Nanotechnol. 10 779-84, Scheible et al 2015 Small 11 5200-5). These advances have naturally triggered the question: What can these DNA nanostructures be used for? One of the leading proposals of use for DNA nanotechnology has been in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of

  7. 59. Cold Spring Harbor symposium on quantitative biology: Molecular genetics of cancer

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    Investigation of the mechanistic aspects of cancer has its roots in the studies on tumor viruses and their effects on cell proliferation, function, and growth. This outstanding progress was well documented in previous Cold Spring Harbor Symposia on Quantitative Biology. In the early to mid 1980s, progress on the development of chromosome mapping strategies and the accumulation of DNA probes that identified polymorphisms, encouraged by the international Human Genome Project, enabled the identification of other genes that contributed to familial inheritance of high susceptibility to specific cancers. This approach was very successful and led to a degree of optimism that one aspect of cancer, the multistep genetic process from early neoplasia to metastatic tumors, was beginning to be understood. It therefore seemed appropriate that the 59th Symposium on Quantitative Biology focus attention on the Molecular Genetics of Cancer. The concept was to combine the exciting progress on the identification of new genetic alterations in human tumor cells with studies on the function of the cancer gene products and how they go awry in tumor cells.

  8. Single molecular biology: coming of age in DNA replication.

    Science.gov (United States)

    Liu, Xiao-Jing; Lou, Hui-Qiang

    2017-09-20

    DNA replication is an essential process of the living organisms. To achieve precise and reliable replication, DNA polymerases play a central role in DNA synthesis. Previous investigations have shown that the average rates of DNA synthesis on the leading and lagging strands in a replisome must be similar to avoid the formation of significant gaps in the nascent strands. The underlying mechanism has been assumed to be coordination between leading- and lagging-strand polymerases. However, Kowalczykowski's lab members recently performed single molecule techniques in E. coli and showed the real-time behavior of a replisome. The leading- and lagging-strand polymerases function stochastically and independently. Furthermore, when a DNA polymerase is paused, the helicase slows down in a self-regulating fail-safe mechanism, akin to a ''dead-man's switch''. Based on the real-time single-molecular observation, the authors propose that leading- and lagging-strand polymerases synthesize DNA stochastically within a Gaussian distribution. Along with the development and application of single-molecule techniques, we will witness a new age of DNA replication and other biological researches.

  9. DNA assembly for plant biology: techniques and tools.

    Science.gov (United States)

    Patron, Nicola J

    2014-06-01

    As the speed and accuracy of genome sequencing improves, there are ever-increasing resources available for the design and construction of synthetic DNA parts. These can be used to engineer plant genomes to produce new functions or to elucidate the function of endogenous sequences. Until recently the assembly of amplified or cloned sequences into large and complex designs was a limiting step in plant synthetic biology and biotechnology. A number of new methods for assembling DNA molecules have been developed in the last few years, several of which have been applied to the production of molecules used to modify plant genomes.

  10. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  11. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

    Science.gov (United States)

    Booth, Michael J.; Marsico, Giovanni; Bachman, Martin; Beraldi, Dario; Balasubramanian, Shankar

    2014-05-01

    Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

  12. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    Science.gov (United States)

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

  13. Toward quantitative fluorescence microscopy with DNA origami nanorulers.

    Science.gov (United States)

    Beater, Susanne; Raab, Mario; Tinnefeld, Philip

    2014-01-01

    The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution.

  14. Quantitative analysis of the DNA distribution on cigarette butt filter paper.

    Science.gov (United States)

    Casey, Lisa; Engen, Sarah; Frank, Greg

    2013-03-01

    The distribution of DNA on the filter paper of smoked cigarette butts was quantitatively mapped using real-time quantitative polymerase chain reaction. The filter papers from smoked cigarette butts collected from indoor and outdoor sources were sliced into equal pieces and the amount of DNA on each slice was determined. This study found that the cigarette butt filter papers sliced parallel to the seam of the cigarette had more uniformly distributed DNA on the slices and in most cases, there was enough DNA on each slice to obtain a complete DNA profile. The perpendicular slices had a less uniform pattern of distribution and some slices did not have enough DNA to obtain an interpretable DNA profile. Cigarette butts found indoors also had more DNA per cigarette on average than cigarette butts found outdoors.

  15. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

    Science.gov (United States)

    Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

    2011-09-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

  16. Quantitative detection of single DNA molecules on DNA tetrahedron decorated substrates.

    Science.gov (United States)

    Wang, Zhenguang; Xue, Qingwang; Tian, Wenzhi; Wang, Lei; Jiang, Wei

    2012-10-01

    A single DNA molecule detection method on DNA tetrahedron decorated substrates has been developed. DNA tetrahedra were introduced onto substrates for both preventing nonspecific adsorption and sensitive recognition of single DNA molecules.

  17. Molecular biology of Homo sapiens: Abstracts of papers presented at the 51st Cold Spring Harbor symposium on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    Watson, J.D.; Siniscalco, M.

    1986-01-01

    This volume contains abstracts of papers presented at the 51st Cold Springs Harbor Symposium on Quantitative Biology. The topic for this meeting was the ''Molecular Biology of Homo sapiens.'' Sessions were entitled Human Gene Map, Human Cancer Genes, Genetic Diagnosis, Human Evolution, Drugs Made Off Human Genes, Receptors, and Gene Therapy. (DT)

  18. Thirty-five years of Tropical biology: a quantitative history

    OpenAIRE

    Monge-Nájera, Julian; Díaz, Lizeth

    2016-01-01

    Citation indices are unappropriated measures of scientific output and impact. For that reason, nonparametric statistics were preferred to analyze 35 years of publication on Tropical biology in the Revista de Biología Tropical. The most frequent subjects are animal taxonomy, human biology -including medicine- ecology and animal behavior. Botany papers are less frequent and mainly deal with morphology and taxonomy. Applied studies are not predominant. In that period, only one case of unethical ...

  19. Quantitation by flow microfluorometry of total cellular DNA in Acanthamoeba

    Energy Technology Data Exchange (ETDEWEB)

    Coulson, P.B.; Tyndall, R.

    1978-01-01

    The DNA content of five speciea of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24 to 48 hr. The trophozoites were harvested, fixed in 70% ethanol (acidified), pretreated with RNase, stained with propidium diiodide, and evaluated for DNA-bound fluorescence. All species tested had DNA values between 2.0 to 5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.

  20. Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yun-bo

    1988-03-01

    This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs.

  1. Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

    Science.gov (United States)

    Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

    2017-02-01

    DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr (+) ][Ntf 2(-) ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

  2. Using Active Learning to Teach Concepts and Methods in Quantitative Biology.

    Science.gov (United States)

    Waldrop, Lindsay D; Adolph, Stephen C; Diniz Behn, Cecilia G; Braley, Emily; Drew, Joshua A; Full, Robert J; Gross, Louis J; Jungck, John A; Kohler, Brynja; Prairie, Jennifer C; Shtylla, Blerta; Miller, Laura A

    2015-11-01

    This article provides a summary of the ideas discussed at the 2015 Annual Meeting of the Society for Integrative and Comparative Biology society-wide symposium on Leading Students and Faculty to Quantitative Biology through Active Learning. It also includes a brief review of the recent advancements in incorporating active learning approaches into quantitative biology classrooms. We begin with an overview of recent literature that shows that active learning can improve students' outcomes in Science, Technology, Engineering and Math Education disciplines. We then discuss how this approach can be particularly useful when teaching topics in quantitative biology. Next, we describe some of the recent initiatives to develop hands-on activities in quantitative biology at both the graduate and the undergraduate levels. Throughout the article we provide resources for educators who wish to integrate active learning and technology into their classrooms.

  3. Modeling Cancer Metastasis using Global, Quantitative and Integrative Network Biology

    DEFF Research Database (Denmark)

    Schoof, Erwin; Erler, Janine

    phosphorylation dynamics in a given biological sample. In Chapter III, we move into Integrative Network Biology, where, by combining two fundamental technologies (MS & NGS), we can obtain more in-depth insights into the links between cellular phenotype and genotype. Article 4 describes the proof...... cancer networks using Network Biology. Technologies key to this, such as Mass Spectrometry (MS), Next-Generation Sequencing (NGS) and High-Content Screening (HCS) are briefly described. In Chapter II, we cover how signaling networks and mutational data can be modeled in order to gain a better...

  4. Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

    National Research Council Canada - National Science Library

    Newman, Michelle; Blyth, Benjamin J; Hussey, Damian J; Jardine, Daniel; Sykes, Pamela J; Ormsby, Rebecca J

    2012-01-01

    We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1...

  5. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    Science.gov (United States)

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  6. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

    Science.gov (United States)

    Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

    2016-02-01

    The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

  7. Insights Into Quantitative Biology: analysis of cellular adaptation

    OpenAIRE

    Agoni, Valentina

    2013-01-01

    In the last years many powerful techniques have emerged to measure protein interactions as well as gene expression. Many progresses have been done since the introduction of these techniques but not toward quantitative analysis of data. In this paper we show how to study cellular adaptation and how to detect cellular subpopulations. Moreover we go deeper in analyzing signal transduction pathways dynamics.

  8. DNA origami-based standards for quantitative fluorescence microscopy.

    Science.gov (United States)

    Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

    2014-01-01

    Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

  9. Mitochondrial DNA deletion analysis: a comparison of PCR quantitative methods.

    Science.gov (United States)

    Hamblet, N S; Castora, F J

    1995-02-15

    The role of mitochondrial DNA (mtDNA) deletions in aging and in neurodegenerative diseases is often determined by measuring the amount of deleted mtDNA in the affected tissue. Upon examining brain autopsy tissue from a 59 year old individual with lung cancer we determined by serial dilution PCR and kinetic PCR that a greater ratio of deleted mtDNA was present in the caudate than in the parietal cortex. However, the magnitude difference for these two brain regions appeared to be technique dependent; by serial dilution PCR the caudate had 10 times more deleted mtDNA than the parietal cortex (0.0141 vs 0.0014) whereas kinetic PCR yielded a 4-fold difference (0.1258 vs 0.0316). These results indicate that although it is valid to compare the amount of deleted mtDNA in normal and diseased tissue and draw conclusions based on relative comparisons within one study, greater caution should be exercised when comparing absolute values from studies using different measurement techniques.

  10. Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

    Science.gov (United States)

    Eychner, Alison M; Lebo, Roberta J; Elkins, Kelly M

    2015-06-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.

  11. Case studies in quantitative biology: Biochemistry on a leash and a single-molecule Hershey-Chase experiment

    Science.gov (United States)

    Van Valen, David

    2011-12-01

    The last 50 years of biological research has seen a marked increase in the amount of quantitative data that describes living systems. This wealth of data provides a unique opportunity to recast the pictorial level descriptions of biological processes in the language of mathematics, with the hope that such an undertaking will lead to deeper insights into the behavior of living systems. To achieve this end, we have undertaken three case studies in physical biology. In the first case study, we used statistical mechanics and polymer physics to construct a simple model that describes how flexible chains of amino acids, referred to as tethers, influence the information processing properties of signaling proteins. In the second case study, we studied the DNA ejection process of phage lambda in vitro. In particular, we used bulk and single-molecule methods to study the control parameters that govern the force and kinematics of the ejection process in vitro. In the last case study, we studied the DNA ejection process of phage lambda in vivo. We developed an assay that allows real-time monitoring of DNA ejection in vivo at the single-molecule level. We also developed a parallel system that allows the simultaneous visualization of both phage capsids and phage DNA at the single-cell level, constituting a true single-molecule Hershey-Chase experiment. The work described in this thesis outlines new tools, both in theory and experiment, that can be used to study biological systems as well as a paradigm that can be employed to mathematicize the cartoons of biology.

  12. Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

    OpenAIRE

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-01-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from th...

  13. [Method validation according to ISO 15189 and SH GTA 04: application for the extraction of DNA and its quantitative evaluation by a spectrophotometric assay].

    Science.gov (United States)

    Harlé, Alexandre; Lion, Maëva; Husson, Marie; Dubois, Cindy; Merlin, Jean-Louis

    2013-01-01

    According to the French legislation on medical biology (January 16th, 2010), all biological laboratories must be accredited according to ISO 15189 for at least 50% of their activities before the end of 2016. The extraction of DNA from a sample of interest, whether solid or liquid is one of the critical steps in molecular biology and specifically in somatic or constitutional genetic. The extracted DNA must meet a number of criteria such quality and also be in sufficient concentration to allow molecular biology assays such as the detection of somatic mutations. This paper describes the validation of the extraction and purification of DNA using chromatographic column extraction and quantitative determination by spectrophotometric assay, according to ISO 15189 and the accreditation technical guide in Human Health SH-GTA-04.

  14. Photon-tissue interaction model for quantitative assessment of biological tissues

    Science.gov (United States)

    Lee, Seung Yup; Lloyd, William R.; Wilson, Robert H.; Chandra, Malavika; McKenna, Barbara; Simeone, Diane; Scheiman, James; Mycek, Mary-Ann

    2014-02-01

    In this study, we describe a direct fit photon-tissue interaction model to quantitatively analyze reflectance spectra of biological tissue samples. The model rapidly extracts biologically-relevant parameters associated with tissue optical scattering and absorption. This model was employed to analyze reflectance spectra acquired from freshly excised human pancreatic pre-cancerous tissues (intraductal papillary mucinous neoplasm (IPMN), a common precursor lesion to pancreatic cancer). Compared to previously reported models, the direct fit model improved fit accuracy and speed. Thus, these results suggest that such models could serve as real-time, quantitative tools to characterize biological tissues assessed with reflectance spectroscopy.

  15. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods

    Directory of Open Access Journals (Sweden)

    Sedlackova Tatiana

    2013-02-01

    Full Text Available Abstract Background Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR. Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

  16. Quantitative Link Between Biological Evolution and Statistical Mechanics

    Science.gov (United States)

    Ray, Tane S.

    A model of evolution called the modified Wright-Fisher model (MWF) is introduced. It is shown to exhibit a second order phase transition, and a quantitative mapping is established between the mean field Ising model and itself. An equation of state and scaling function are derived for the MWF from the steady state solution of the governing quasispecies equations. The critical exponents are identical to those of the mean-field Ising model. Simulation data for the MWF on a two-dimensional square lattice show good evidence for a critical point. The susceptibility exponent is estimated and is found, within the uncertainty of the simulation data, to be equal to that of the two-dimensional Ising model, suggesting that the two models are in the same universality class.

  17. A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.

    Science.gov (United States)

    Concannon, Sean P; Wimberley, P Brett; Workman, Wesley E

    2011-01-01

    Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550-554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3-3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.

  18. A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

    Science.gov (United States)

    Claus, Rainer; Wilop, Stefan; Hielscher, Thomas; Sonnet, Miriam; Dahl, Edgar; Galm, Oliver; Jost, Edgar; Plass, Christoph

    2012-01-01

    Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly. PMID:22647397

  19. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real‐time PCR

    National Research Council Canada - National Science Library

    Desneux, Jérémy; Pourcher, Anne‐Marie

    2014-01-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil ® , PowerFecal ® , NucleoSpin...

  20. Conversion of cDNA differential display results (DDRT-PCR into quantitative transcription profiles

    Directory of Open Access Journals (Sweden)

    Koopmann Birger

    2005-04-01

    Full Text Available Abstract Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii intensity of bands is determined by densitometry, (iii densitometric values are normalized, and (iv intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

  1. DNA intercalation by ethidium bromide: A quantitative binding study using DNA stretching and force-induced melting

    Science.gov (United States)

    Williams, Mark C.; Rouzina, Ioulia

    2005-03-01

    The interactions between single DNA molecules and the non- covalent binding agent ethidium bromide are investigated using an optical tweezers instrument and the effects of this intercalator on the structure and mechanical stability of DNA molecules are quantitatively analyzed using our model of force- induced melting. The DNA force-extension cycles in the presence and absence of drug are recorded. It is found that the drug binds preferentially to double-stranded DNA and stabilizes the double helix. There is clear evidence of the force induced melting transition at low concentrations of drug, while at higher concentrations the drug is able to prevent the melting transition. The DNA contour length is obtained as a function of ligand concentration directly from the stretching curves. From this data we obtain the complete ethidium bromide dsDNA binding isotherm, which is used to find the binding constant and the binding site size of the intercalator. Out data also allows us to quantify directly the effect of ethidium bromide on the free energy of the helix-coil transition in dsDNA. This single molecule study brings new insights into the molecular mechanisms which drive drug-DNA complex formation.

  2. Sender-receiver systems and applying information theory for quantitative synthetic biology.

    Science.gov (United States)

    Barcena Menendez, Diego; Senthivel, Vivek Raj; Isalan, Mark

    2015-02-01

    Sender-receiver (S-R) systems abound in biology, with communication systems sending information in various forms. Information theory provides a quantitative basis for analysing these processes and is being applied to study natural genetic, enzymatic and neural networks. Recent advances in synthetic biology are providing us with a wealth of artificial S-R systems, giving us quantitative control over networks with a finite number of well-characterised components. Combining the two approaches can help to predict how to maximise signalling robustness, and will allow us to make increasingly complex biological computers. Ultimately, pushing the boundaries of synthetic biology will require moving beyond engineering the flow of information and towards building more sophisticated circuits that interpret biological meaning.

  3. Sender–receiver systems and applying information theory for quantitative synthetic biology

    Science.gov (United States)

    Barcena Menendez, Diego; Senthivel, Vivek Raj; Isalan, Mark

    2015-01-01

    Sender–receiver (S–R) systems abound in biology, with communication systems sending information in various forms. Information theory provides a quantitative basis for analysing these processes and is being applied to study natural genetic, enzymatic and neural networks. Recent advances in synthetic biology are providing us with a wealth of artificial S–R systems, giving us quantitative control over networks with a finite number of well-characterised components. Combining the two approaches can help to predict how to maximise signalling robustness, and will allow us to make increasingly complex biological computers. Ultimately, pushing the boundaries of synthetic biology will require moving beyond engineering the flow of information and towards building more sophisticated circuits that interpret biological meaning. PMID:25282688

  4. Development and Assessment of Modules to Integrate Quantitative Skills in Introductory Biology Courses.

    Science.gov (United States)

    Hoffman, Kathleen; Leupen, Sarah; Dowell, Kathy; Kephart, Kerrie; Leips, Jeff

    2016-01-01

    Redesigning undergraduate biology courses to integrate quantitative reasoning and skill development is critical to prepare students for careers in modern medicine and scientific research. In this paper, we report on the development, implementation, and assessment of stand-alone modules that integrate quantitative reasoning into introductory biology courses. Modules are designed to improve skills in quantitative numeracy, interpreting data sets using visual tools, and making inferences about biological phenomena using mathematical/statistical models. We also examine demographic/background data that predict student improvement in these skills through exposure to these modules. We carried out pre/postassessment tests across four semesters and used student interviews in one semester to examine how students at different levels approached quantitative problems. We found that students improved in all skills in most semesters, although there was variation in the degree of improvement among skills from semester to semester. One demographic variable, transfer status, stood out as a major predictor of the degree to which students improved (transfer students achieved much lower gains every semester, despite the fact that pretest scores in each focus area were similar between transfer and nontransfer students). We propose that increased exposure to quantitative skill development in biology courses is effective at building competency in quantitative reasoning.

  5. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology

    Directory of Open Access Journals (Sweden)

    Francesca Grasso

    2015-08-01

    Full Text Available Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.

  6. SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena.

    Science.gov (United States)

    Tohsato, Yukako; Ho, Kenneth H L; Kyoda, Koji; Onami, Shuichi

    2016-11-15

    Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. SSBD is accessible at http://ssbd.qbic.riken.jp CONTACT: sonami@riken.jp. © The Author 2016. Published by Oxford University Press.

  7. Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

    Science.gov (United States)

    Aedo, Sandra

    2012-01-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA. PMID:22869559

  8. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    Science.gov (United States)

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

  9. Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA.

    Science.gov (United States)

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-11-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

  10. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  11. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    Science.gov (United States)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  12. Sensitive, simultaneous quantitation of two unlabeled DNA targets using a magnetic nanoparticle-enzyme sandwich assay.

    Science.gov (United States)

    Zhang, Yue; Pilapong, Chalermchai; Guo, Yuan; Ling, Zhenlian; Cespedes, Oscar; Quirke, Philip; Zhou, Dejian

    2013-10-01

    We report herein the development of a simple, sensitive colorimetric magnetic nanoparticle (MNP)-enzyme-based DNA sandwich assay that is suitable for simultaneous, label-free quantitation of two DNA targets down to 50 fM level. It can also effectively discriminate single-nucleotide polymorphisms (SNPs) in genes associated with human cancers (KRAS codon 12/13 SNPs). This assay uses a pair of specific DNA probes, one being covalently conjugated to an MNP for target capture and the other being linked to an enzyme for signal amplification, to sandwich a DNA target, allowing for convenient magnetic separation and subsequent efficient enzymatic signal amplification for high sensitivity. Careful optimization of the MNP surfaces and assay conditions greatly reduced the background, allowing for sensitive, specific detection of as little as 5 amol (50 fM in 100 μL) of target DNA. Moreover, this sensor is robust, it can effectively discriminate cancer-specific SNPs against the wild-type noncancer target, and it works efficiently in 10% human serum. Furthermore, this sensor can simultaneously quantitate two different DNA targets by using two pairs of unique capture- and signal-DNA probes specific for each target. This general, simple, and sensitive DNA sensor appears to be well-suited for a wide range of genetics-based biosensing and diagnostic applications.

  13. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    Science.gov (United States)

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  14. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

    Directory of Open Access Journals (Sweden)

    Ana Lisa do Vale Gomes

    2006-10-01

    Full Text Available This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA. The efficiency was 0.99 and the correlation coefficient (R² was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC. The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

  15. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    Science.gov (United States)

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

  16. Quantitative Analysis of the Trends Exhibited by the Three Interdisciplinary Biological Sciences: Biophysics, Bioinformatics, and Systems Biology.

    Science.gov (United States)

    Kang, Jonghoon; Park, Seyeon; Venkat, Aarya; Gopinath, Adarsh

    2015-12-01

    New interdisciplinary biological sciences like bioinformatics, biophysics, and systems biology have become increasingly relevant in modern science. Many papers have suggested the importance of adding these subjects, particularly bioinformatics, to an undergraduate curriculum; however, most of their assertions have relied on qualitative arguments. In this paper, we will show our metadata analysis of a scientific literature database (PubMed) that quantitatively describes the importance of the subjects of bioinformatics, systems biology, and biophysics as compared with a well-established interdisciplinary subject, biochemistry. Specifically, we found that the development of each subject assessed by its publication volume was well described by a set of simple nonlinear equations, allowing us to characterize them quantitatively. Bioinformatics, which had the highest ratio of publications produced, was predicted to grow between 77% and 93% by 2025 according to the model. Due to the large number of publications produced in bioinformatics, which nearly matches the number published in biochemistry, it can be inferred that bioinformatics is almost equal in significance to biochemistry. Based on our analysis, we suggest that bioinformatics be added to the standard biology undergraduate curriculum. Adding this course to an undergraduate curriculum will better prepare students for future research in biology.

  17. Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA

    Institute of Scientific and Technical Information of China (English)

    Ya-Xi Chen; Ai-Long Huang; Zhen-Yuan Qi; Shu-Hua Guo

    2004-01-01

    AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR).METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography,and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigeninlabeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigeninlabeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed.RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100%compared with digoxigenin-labeled DHBV DNA probe assay.After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis.Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle.The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficientof the results was (0.97, P<0.01) between fluorescent qPCRand dot-blot hybridization.CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be

  18. Quantitative analysis of reptation of partially extended DNA in sub-30 nm nanoslits

    CERN Document Server

    Yeh, Jia-Wei; Taloni, Alessandro; Chen, Yeng-Long; Chou, Chia-Fu

    2015-01-01

    We observed reptation of single DNA molecules in fused silica nanoslits of sub-30 nm height. The reptation behavior and the effect of confinement are quantitatively characterized using orientation correlation and transverse fluctuation analysis. We show tube-like polymer motion arises for a tense polymer under strong quasi-2D confinement and interaction with surface- passivating polyvinylpyrrolidone (PVP) molecules in nanoslits, while etching- induced device surface roughness, chip bonding materials and DNA-intercalated dye-surface interaction, play minor roles. These findings have strong implications for the effect of surface modification in nanofluidic systems with potential applications for single molecule DNA analysis.

  19. Prospects and challenges of quantitative phase imaging in tumor cell biology

    Science.gov (United States)

    Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

    2016-03-01

    Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

  20. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

    Science.gov (United States)

    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

  1. A census of cells in time: quantitative genetics meets developmental biology.

    Science.gov (United States)

    Chitwood, Daniel H; Sinha, Neelima R

    2013-02-01

    Quantitative genetics has become a popular method for determining the genetic basis of natural variation. Combined with genomic methods, it provides a tool for discerning the genetic basis of gene expression. So-called genetical genomics approaches yield a wealth of genomic information, but by necessity, because of cost and time, fail to resolve the differences between organs, tissues, and/or cell types. Similarly, quantitative approaches in development that might potentially address these issues are seldom applied to quantitative genetics. We discuss recent advances in cell type-specific isolation methods, the quantitative analysis of phenotype, and developmental modeling that are compatible with quantitative genetics and, with time, promise to bridge the gap between these two powerful disciplines yielding unprecedented biological insight.

  2. Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

    Directory of Open Access Journals (Sweden)

    Bogachev Sergei S

    2006-10-01

    Full Text Available Abstract Background The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell. Results The existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7 cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction was assessed. The extracellular DNA delivered to the cell is characterized quantitatively. Conclusion It is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.

  3. Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David G. J. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology; McKnight, Timothy E. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Engineering Science and Technology Division; Melechko, Anatoli V. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Simpson, Michael L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Sayler, Gary S. [Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology

    2006-12-08

    Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. We used polymerase chain reaction (PCR) and in vitro transcription (IVT) to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. In conclusion,this investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

  4. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    Science.gov (United States)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  5. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions

    Science.gov (United States)

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-01

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

  6. The DNA sequence and biology of human chromosome 19.

    Science.gov (United States)

    Grimwood, Jane; Gordon, Laurie A; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Isaac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E; Pennacchio, Len A; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S; Myers, Richard M; Rubin, Edward M; Lucas, Susan M

    2004-04-01

    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 G + C 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 hypercholesterolaemia 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.

  7. 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.

  8. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, Jane; Gordon, Laurie A.; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A.; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C.; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M.; Glavina, Tijana; Gomez, Maria; Gonzales, Eldelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Issac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P.; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S.; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E.; Pennacchio, Len A.; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S.; Myers, Richard M.; Rubin, Edward M.; Lucas, Susan M.

    2003-09-15

    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 G1C 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 percent 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 hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25 percent 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, a nd segments of coding and non-coding conservation with the distant fish species Takifugu.

  9. Modeling optical behavior of birefringent biological tissues for evaluation of quantitative polarized light microscopy

    NARCIS (Netherlands)

    Turnhout, van M.C.; Kranenbarg, S.; Leeuwen, van J.L.

    2009-01-01

    Quantitative polarized light microscopy (qPLM) is a popular tool for the investigation of birefringent architectures in biological tissues. Collagen, the most abundant protein in mammals, is such a birefringent material. Interpretation of results of qPLM in terms of collagen network architecture and

  10. Cold Spring Harbor symposia on quantitative biology. Volume 54, Immunological recognition

    Energy Technology Data Exchange (ETDEWEB)

    1989-12-31

    This volume contains the second part of the proceedings of the 53rd Cold Springs Harbor Symposium on Quantitative Biology. This years topic was Immune Recognition. This volume, part 2, contains papers prepared by presenters for two sessions entitled Signals for Lymphocyte Activation, Proliferation, and Adhesion, and entitled Tolerance and Self Recognition. (DT)

  11. Cold Spring Harbor symposia on quantitative biology. Volume 54, Immunological recognition

    Energy Technology Data Exchange (ETDEWEB)

    1989-12-31

    This volume contains the first part of the proceeding of the 53rd Cold Springs Harbor Symposium on Quantitative Biology. This years topic was Immune Recognition. Part 1, this volume, contains papers prepared by presenters of the sessions entitled Introduction, Lymphocyte Development and Receptor Selection, and Recognition by Antibodies, Antigen Recognition by T cells. (DT)

  12. Studying Biology to Understand Risk: Dosimetry Models and Quantitative Adverse Outcome Pathways

    Science.gov (United States)

    Confidence in the quantitative prediction of risk is increased when the prediction is based to as great an extent as possible on the relevant biological factors that constitute the pathway from exposure to adverse outcome. With the first examples now over 40 years old, physiologi...

  13. Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

    Science.gov (United States)

    Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

    2016-01-07

    Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

  14. Fuzzy Logic as a Computational Tool for Quantitative Modelling of Biological Systems with Uncertain Kinetic Data.

    Science.gov (United States)

    Bordon, Jure; Moskon, Miha; Zimic, Nikolaj; Mraz, Miha

    2015-01-01

    Quantitative modelling of biological systems has become an indispensable computational approach in the design of novel and analysis of existing biological systems. However, kinetic data that describe the system's dynamics need to be known in order to obtain relevant results with the conventional modelling techniques. These data are often hard or even impossible to obtain. Here, we present a quantitative fuzzy logic modelling approach that is able to cope with unknown kinetic data and thus produce relevant results even though kinetic data are incomplete or only vaguely defined. Moreover, the approach can be used in the combination with the existing state-of-the-art quantitative modelling techniques only in certain parts of the system, i.e., where kinetic data are missing. The case study of the approach proposed here is performed on the model of three-gene repressilator.

  15. High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro.

    Science.gov (United States)

    Sil, Payel; Yoo, Dae-Goon; Floyd, Madison; Gingerich, Aaron; Rada, Balazs

    2016-06-18

    Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

  16. Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA.

    Science.gov (United States)

    Jiao, Jingjing; Kang, Jing X; Tan, Rui; Wang, Jingdong; Zhang, Yu

    2012-04-01

    In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.

  17. Dynamical model for biological functions of DNA molecules

    Institute of Scientific and Technical Information of China (English)

    PANGXiao-fengI; YANGYao

    2004-01-01

    We proposed a dynamic model of DNA to study its nonlinear excitation and duplication and transcription in the basis of molecular structure and changes of conformation of DNA under influence of bioenergy.

  18. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  19. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  20. Quantitation of Genital Herpes Virus DNA by Polymerase Chain Reaction and ELISA

    Institute of Scientific and Technical Information of China (English)

    CHENG Peihua(程培华)

    2002-01-01

    Objective:To detect and quantitate genital herpes simplex virus (HSV) DNA in specimens from 100 patients clinically diagnosed with genital herpes.Methods: Polymerase Chain Reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used with a standard curve of DNA copies of HSV as quantitative contrast.Results: Ninety-three cases were confirmed HSV positive and 7 cases were found to be negative. There were 58 cases of HSV-2 (62.4%) and 35 cases of HSV-1 (37.6%) among the 93 positive cases. The number of DNA plasmids ranged from 115 to 1.1×105 per 250μL among the 93 positive samples (mean =7.1×104/250μL). The number of HSV DNA plasmids ranged from 136 to 1.1×105 copies per 250μL (mean =7.6×104) among those with HSV-2, and 115 to 9.4×104 per 250μL (mean =6.3×104) among those with HSV-1. Meanwhile 10μL of extracted and dissolved DNA randomly taken from 8 each of HSV-2 and HSV-1 samples were tested. The number of HSV-2 DNA plasmids ranged from 35 copies to 2.7×104 (Mean =1.8×104) and the number of HSV-1 DNA ranged from 29 to 2.5×104 (Mean = 1.6×104). In the 7 negative cases, the quantity of HSV plasmids was zero.Conclusion: The sensitivity of ELISA quantitation (93%) is equal to that of Southern blot. The sensitivity of PCR for diagnosis is 91%, and 88% for PCR typing.

  1. Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP.

    Directory of Open Access Journals (Sweden)

    Femke L Groeneweg

    Full Text Available Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR and the mineralocorticoid receptor (MR, two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s and the other half for longer time periods (∼ 2.3 s. A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤ 1 ms interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

  2. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    Energy Technology Data Exchange (ETDEWEB)

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  3. Recent progress in quantitative analysis of DNA adducts of nephrotoxin aristolochic acid

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Aristolochic acid (AA), a mixture of structure-related nitrophenanthrene carboxylic acid derivatives derived from Aristolochia spp, is associated with nephrotoxin and carcinogen. AA-DNA adducts induced by reductive metabolic activation of AA were detected in tissues of animals and in patients exposed to AA. The DNA adducts were generally used as biomarkers in toxicological study of AA. In this short review, quantitative analysis of AA-DNA adducts in various in vitro and in vivo systems by using 32P-postlabelling assay, HPLC-UV, HPLC-radiation monitor, HPLC-FLD, HPLC-ESI/MS and UPLC-MS/MS methods is discussed. The distribution of AA-DNA adducts in various tissues is also summarized.

  4. Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    Nai-Zhong XIAO; Lei BA; Preben Bach HOLM; Xing-Zhi WANG; Steve BOWRA

    2005-01-01

    The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived α-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of α-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

  5. Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

    Directory of Open Access Journals (Sweden)

    Martínez-Díaz Rafael A

    2001-01-01

    Full Text Available Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

  6. Research development of residual DNA analysis techniques in biological products%生物制品残留DNA分析技术的研究进展

    Institute of Scientific and Technical Information of China (English)

    吴浩飞

    2013-01-01

    生物制品中残留DNA可能具有感染性和致癌性,因此生物制品的纯化过程需要验证,尽可能将产品中残留DNA的水平降到最低.厂家有必要显示DNA去除过程,并有适当定量分析方法检测残留DNA的含量.当前DNA定量分析所采用的最普遍方法是定量PCR(quantitative real time polymerase chain reaction,Q-PCR).此文就残留DNA的感染性、致癌性、免疫原性等潜在风险以及残留DNA分析技术的最新进展做一综述.%The residual DNA (rDNA) in biological products may be potentially infectious or oncogenic,so purification processes need to be validated for confirming its clearance.It is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm rDNA levels in the final products using an appropriately specific and quantitative analytical method.The most common methodology for rDNA quantitation used currently is a quantitative realtime polymerase chain reaction.In this paper,potential risks of infectivity,oncogenicity,and immunogenicity of rDNA and recent progress in rDNA analysis techniques are reviewed.

  7. Context influences on TALE-DNA binding revealed by quantitative profiling.

    Science.gov (United States)

    Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

    2015-06-11

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

  8. Development and validation of InnoQuant(®) HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

    Science.gov (United States)

    Loftus, Andrew; Murphy, Gina; Brown, Hiromi; Montgomery, Anne; Tabak, Jonathan; Baus, James; Carroll, Marion; Green, André; Sikka, Suresh; Sinha, Sudhir

    2017-07-01

    The development and validation of InnoQuant(®) HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow

  9. Gender, Math Confidence, and Grit: Relationships with Quantitative Skills and Performance in an Undergraduate Biology Course.

    Science.gov (United States)

    Flanagan, K M; Einarson, J

    2017-01-01

    In a world filled with big data, mathematical models, and statistics, the development of strong quantitative skills is becoming increasingly critical for modern biologists. Teachers in this field must understand how students acquire quantitative skills and explore barriers experienced by students when developing these skills. In this study, we examine the interrelationships among gender, grit, and math confidence for student performance on a pre-post quantitative skills assessment and overall performance in an undergraduate biology course. Here, we show that females significantly underperformed relative to males on a quantitative skills assessment at the start of term. However, females showed significantly higher gains over the semester, such that the gender gap in performance was nearly eliminated by the end of the semester. Math confidence plays an important role in the performance on both the pre and post quantitative skills assessments and overall performance in the course. The effect of grit on student performance, however, is mediated by a student's math confidence; as math confidence increases, the positive effect of grit decreases. Consequently, the positive impact of a student's grittiness is observed most strongly for those students with low math confidence. We also found grit to be positively associated with the midterm score and the final grade in the course. Given the relationships established in this study among gender, grit, and math confidence, we provide "instructor actions" from the literature that can be applied in the classroom to promote the development of quantitative skills in light of our findings. © 2017 K. M. Flanagan and J. Einarson. CBE—Life Sciences Education © 2017 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http

  10. Quantitative terahertz time-domain spectroscopy and analysis in chemistry and biology

    DEFF Research Database (Denmark)

    Jepsen, Peter Uhd

    2005-01-01

    crystals and biological material. In order to obtain quantitative results great care in the analysis of the experimental data is required. I will discuss common pitfalls in the analysis of THz-TDS data as well as the influence of electronic and laser noise on the results of a THz-TDS experiment.......I will describe how Terahertz Time-Domain Spectroscopy (THz-TDS) can be used for quantitative, broadband spectroscopy in the far-infrared spectral region. Thz-TDS is sensitive to long-range, non-covalent interactions in the condensed phase, for instance intermolecular hydrogen bonding in molecular...

  11. Designer cantilevers for even more accurate quantitative measurements of biological systems with multifrequency AFM

    Science.gov (United States)

    Contera, S.

    2016-04-01

    Multifrequency excitation/monitoring of cantilevers has made it possible both to achieve fast, relatively simple, nanometre-resolution quantitative mapping of mechanical of biological systems in solution using atomic force microscopy (AFM), and single molecule resolution detection by nanomechanical biosensors. A recent paper by Penedo et al [2015 Nanotechnology 26 485706] has made a significant contribution by developing simple methods to improve the signal to noise ratio in liquid environments, by selectively enhancing cantilever modes, which will lead to even more accurate quantitative measurements.

  12. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera Salicio, Susanna; Busk, Peter K.

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...... settings. RESULTS: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...

  13. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera, Susanna; Busk, Peter Kamp

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... settings. Results We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...

  14. Quantitative radiommunoassay for DNA-binding antibodies. [Iodine 131, Iodine 125

    Energy Technology Data Exchange (ETDEWEB)

    Smith, L.H.; Guyer, R.L.; Minami, R.M.; Teplitz, R.L.

    1981-09-01

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (/sup 125/I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE.

  15. A quantitative model of human DNA base excision repair. I. mechanistic insights

    OpenAIRE

    Sokhansanj, Bahrad A.; Rodrigue, Garry R.; Fitch, J. Patrick; David M Wilson

    2002-01-01

    Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts conside...

  16. Gender, Math Confidence, and Grit: Relationships with Quantitative Skills and Performance in an Undergraduate Biology Course

    Science.gov (United States)

    Flanagan, K. M.; Einarson, J.

    2017-01-01

    In a world filled with big data, mathematical models, and statistics, the development of strong quantitative skills is becoming increasingly critical for modern biologists. Teachers in this field must understand how students acquire quantitative skills and explore barriers experienced by students when developing these skills. In this study, we examine the interrelationships among gender, grit, and math confidence for student performance on a pre–post quantitative skills assessment and overall performance in an undergraduate biology course. Here, we show that females significantly underperformed relative to males on a quantitative skills assessment at the start of term. However, females showed significantly higher gains over the semester, such that the gender gap in performance was nearly eliminated by the end of the semester. Math confidence plays an important role in the performance on both the pre and post quantitative skills assessments and overall performance in the course. The effect of grit on student performance, however, is mediated by a student’s math confidence; as math confidence increases, the positive effect of grit decreases. Consequently, the positive impact of a student’s grittiness is observed most strongly for those students with low math confidence. We also found grit to be positively associated with the midterm score and the final grade in the course. Given the relationships established in this study among gender, grit, and math confidence, we provide “instructor actions” from the literature that can be applied in the classroom to promote the development of quantitative skills in light of our findings. PMID:28798209

  17. Detection of clustered DNA lesions: Biological and clinical applications

    Institute of Scientific and Technical Information of China (English)

    Alexandros; Georgakilas

    2011-01-01

    Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.

  18. Molecular biology. Shielding broken DNA for a quick fix

    DEFF Research Database (Denmark)

    Lukas, Jiri; Lukas, Claudia

    2013-01-01

    A fast-acting DNA repair mechanism involves a protein complex that blocks an alternative process that requires a cell to wait for repair.......A fast-acting DNA repair mechanism involves a protein complex that blocks an alternative process that requires a cell to wait for repair....

  19. Delineating the DNA damage response using systems biology approaches

    NARCIS (Netherlands)

    Stechow, Louise von

    2013-01-01

    Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted

  20. Biological and environmental conditionings for a sperm DNA fragmentation.

    Science.gov (United States)

    Bojar, Iwona; Witczak, Mariusz; Wdowiak, Artur

    2013-01-01

    The objective of the presented study was determination of the effect of selected agents on sperm DNA fragmentation--superoxide dismutase in seminal plasma, the patients' age, and burdening with the tobacco smoking habit. An attempt was also undertaken to evaluate the effect of DNA fragmentation on the effectiveness of infertility treatment. The study covered 186 men who received treatment due to infertility. The database and statistical analyses were performed using computer software STATISTICA 7.1. A relationship was observed between sperm DNA fragmentation and superoxide dismutase activity, the higher the SOD activity, the lower the percentage of sperm fragmentation (rs=-0.324; P=0.000; r = -0.2110). A statistical relationship was found between sperm DNA fragmentation and the percentage of pregnancies obtained during the first year of treatment--patients with the lower DFI more frequently became fathers during the first year of trying, compared to the remainder (t=2.51; P=0.013). A statistically significant relationship was confirmed (rs=-0.370; P=0.000) consisting in an increase in the DFI with respondents' age. No significant differences were noted between the DFI and the tobacco smoking habit (Chi2=0.29; P=0.926). The percentage of sperm DNA fragmentation was inversely proportional to superoxide dismutase activity in seminal plasma. DNA fragmentation becomes intensified with patients' age. Cigarette smoking has no effect on sperm DNA fragmentation. DNA fragmentation exerts an effect on the effectiveness of infertility treatment.

  1. An Improved DNA Extraction Method for Efficient and Quantitative Recovery of Phytoplankton Diversity in Natural Assemblages.

    Directory of Open Access Journals (Sweden)

    Jian Yuan

    Full Text Available Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eukaryotes, including Archaea, Amoebozoa, Chlorophyta, Ciliphora, Bacillariophyta, Dinophyta, Fungi, Metazoa, etc. The appearance of thecate dinoflagellates, thin-walled phytoplankton and "naked" unicellular organisms indicates that our method could obtain the intact DNA of organisms with different cell coverings. All the results demonstrate that our method is useful for DNA extraction of phytoplankton and environmental surveys of their diversity and abundance.

  2. An Improved DNA Extraction Method for Efficient and Quantitative Recovery of Phytoplankton Diversity in Natural Assemblages

    Science.gov (United States)

    Yuan, Jian; Li, Meizhen; Lin, Senjie

    2015-01-01

    Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds) in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eukaryotes, including Archaea, Amoebozoa, Chlorophyta, Ciliphora, Bacillariophyta, Dinophyta, Fungi, Metazoa, etc. The appearance of thecate dinoflagellates, thin-walled phytoplankton and “naked” unicellular organisms indicates that our method could obtain the intact DNA of organisms with different cell coverings. All the results demonstrate that our method is useful for DNA extraction of phytoplankton and environmental surveys of their diversity and abundance. PMID:26218575

  3. Functional genomics bridges the gap between quantitative genetics and molecular biology.

    Science.gov (United States)

    Lappalainen, Tuuli

    2015-10-01

    Deep characterization of molecular function of genetic variants in the human genome is becoming increasingly important for understanding genetic associations to disease and for learning to read the regulatory code of the genome. In this paper, I discuss how recent advances in both quantitative genetics and molecular biology have contributed to understanding functional effects of genetic variants, lessons learned from eQTL studies, and future challenges in this field.

  4. Extension of nano-confined DNA: quantitative comparison between experiment and theory

    CERN Document Server

    Iarko, V; Nyberg, L K; Müller, V; Fritzsche, J; Ambjörnsson, T; Beech, J P; Tegenfeldt, J O; Mehlig, K; Westerlund, F; Mehlig, B

    2015-01-01

    The extension of DNA confined to nanochannels has been studied intensively and in detail. Yet quantitative comparisons between experiments and model calculations are difficult because most theoretical predictions involve undetermined prefactors, and because the model parameters (contour length, Kuhn length, and effective width) are difficult to compute reliably, leading to a substantial uncertainties. Here we use a recent asymptotically exact theory for the DNA extension in the "extended de Gennes regime" that allows to determine the model parameters by comparing experimental results with theory. We obtained new experimental results for this purpose, for the mean DNA extension and its standard deviation, varying the channel geometry, dye intercalation ratio, and ionic buffer strength. The experimental results agree very well with theory at high ionic strengths, indicating that the model parameters are reliable. At low ionic strengths the agreement is less good. We discuss possible reasons. Our approach allows...

  5. Experimental genomics: The application of DNA microarrays in cellular and molecular biology studies

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellular and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quant itative fashion. DNA microarrays can be used to measure levels of gene expressio n for tens of thousands of gene simultaneously and take advantage of all availab le sequence information for experimental design and data interpretation in pursu it of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a catalogue of all the genes and informati on about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in genome and gene function analysis, gene expression studies, biological signal and defense system, cell cyclereg ulation, mechanism of transcriptional regulation, proteomics, and the functional ity of food component.

  6. How-to-Do-It: Teaching Recombinant DNA Technology in High School Biology Courses.

    Science.gov (United States)

    Dixon, Linda

    1988-01-01

    Reports on the teaching of recombinant DNA technology in high school biology courses. Explains reactions of the public, students, and colleagues to the molecular genetics unit. Indicates equipment, curricular materials, training, workshops, and availability. (RT)

  7. Quantitation of human parvovirus B19 DNA in erythema infectiosum and aplastic crisis.

    Science.gov (United States)

    Ishikawa, Aki; Yoto, Yuko; Tsugawa, Takeshi; Tsutsumi, Hiroyuki

    2014-12-01

    Several publications concerning the methods of real-time PCR for human parvovirus B19 (B19V) have appeared and some case reports mention B19V DNA loads. However, no large-scale study quantitating levels of B19V DNA in common or representative B19V manifestations such as erythema infectiosum and aplastic crisis has been performed. Consequently, using the TaqMan PCR assay, the B19V load in a large sample of subjects with erythema infectiosum or aplastic crisis was quantitated. Sixty-five subjects in the acute phase of erythema infectiosum were involved, and in addition 22 serum samples from seven subjects with B19V-associated aplastic crisis complicating chronic hemolytic anemia were also analyzed. In the acute phase of erythema infectiosum the median B19V DNA load in the serum samples from the acute phase of erythema infectiosum was 7.63 × 10(5)  genomes/ml, (range from 4.48 × 10(3) to 8.31 × 10(6)  genomes/ml). The serum B19V DNA load during the acute phase of aplastic crisis complicating chronic hemolytic anemia was extremely high, that is 10(10) -10(13)  genomes/ml, and decreased gradually to around 10(5) genomes/ml over 1-2 months. Although all subjects followed an almost uniform and typical clinical course of erythema infectiosum, there was a large individual variation of B19V DNA loads, that is differences of over 1,000 times. Extremely high B19V loads were observed in subjects with aplastic crisis. This study is the first large scale report of studies of the B19V DNA loads in subjects with erythema infectiosum and aplastic crisis, the most common and significant clinical manifestations by B19V infections. © 2014 Wiley Periodicals, Inc.

  8. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  9. 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.

  10. Cell and molecular biology of DNA methyltransferase 1.

    Science.gov (United States)

    Mohan, K Naga; Chaillet, J Richard

    2013-01-01

    The DNA cytosine methyltransferase 1 (DNMT1) is a ubiquitous nuclear enzyme that catalyzes the well-established reaction of placing methyl groups on the unmethylated cytosines in methyl-CpG:CpG base pairs in the hemimethylated DNA formed by methylated parent and unmethylated daughter strands. This activity regenerates fully methylated methyl-CpG:methyl-CpG pairs. Despite the straightforward nature of its catalytic activity, detailed biochemical, genetic, and developmental studies revealed intricate details of the central regulatory role of DNMT1 in governing the epigenetic makeup of the nuclear genome. DNMT1 mediates demethylation and also participates in seemingly wide cellular functions unrelated to maintenance DNA methylation. This review brings together mechanistic details of maintenance methylation by DNMT1, its regulation at transcriptional and posttranscriptional levels, and the seemingly unexpected functions of DNMT1 in the context of DNA methylation which is central to epigenetic changes that occur during development and the process of cell differentiation.

  11. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  12. A robust GC-MS method for the quantitation of fatty acids in biological systems.

    Science.gov (United States)

    Jayasinghe, Nirupama Samanmalie; Dias, Daniel Anthony

    2013-01-01

    Fatty acids (FAs) are involved in a wide range of functions in biological systems. It is important to measure the exact amount of fatty acids in biological matrices in order to determine the level of fatty acids and understand the role they play. The ability to quantify fatty acids in various systems, especially plant species and microbes has recently paved the way to the mass production of pharmaceuticals and energy substitutes including biodiesel. This chapter describes an efficient method to quantify the total fatty acids (TFAs) in biological systems using gas chromatography-mass spectrometry (GC-MS) and a commercially available standard mix of fatty acid methyl esters (FAMEs) using a step-by-step methodology to setup a quantitation method using the Agilent Chemstation software.

  13. Preparation of Biological Samples Containing Metoprolol and Bisoprolol for Applying Methods for Quantitative Analysis

    Directory of Open Access Journals (Sweden)

    Corina Mahu Ştefania

    2015-12-01

    Full Text Available Arterial hypertension is a complex disease with many serious complications, representing a leading cause of mortality. Selective beta-blockers such as metoprolol and bisoprolol are frequently used in the management of hypertension. Numerous analytical methods have been developed for the determination of these substances in biological fluids, such as liquid chromatography coupled with mass spectrometry, gas chromatography coupled with mass spectrometry, high performance liquid chromatography. Due to the complex composition of biological fluids a biological sample pre-treatment before the use of the method for quantitative determination is required in order to remove proteins and potential interferences. The most commonly used methods for processing biological samples containing metoprolol and bisoprolol were identified through a thorough literature search using PubMed, ScienceDirect, and Willey Journals databases. Articles published between years 2005-2015 were reviewed. Protein precipitation, liquid-liquid extraction and solid phase extraction are the main techniques for the extraction of these drugs from plasma, serum, whole blood and urine samples. In addition, numerous other techniques have been developed for the preparation of biological samples, such as dispersive liquid-liquid microextraction, carrier-mediated liquid phase microextraction, hollow fiber-protected liquid phase microextraction, on-line molecularly imprinted solid phase extraction. The analysis of metoprolol and bisoprolol in human plasma, urine and other biological fluids provides important information in clinical and toxicological trials, thus requiring the application of appropriate extraction techniques for the detection of these antihypertensive substances at nanogram and picogram levels.

  14. Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

    Directory of Open Access Journals (Sweden)

    Duborjal Hervé

    2008-02-01

    Full Text Available Abstract Background Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus. Results Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population. Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1. Conclusion This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized

  15. Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique.

    Science.gov (United States)

    Jelinek, Jaroslav; Liang, Shoudan; Lu, Yue; He, Rong; Ramagli, Louis S; Shpall, Elizabeth J; Estecio, Marcos R H; Issa, Jean-Pierre J

    2012-12-01

    Genome wide analysis of DNA methylation provides important information in a variety of diseases, including cancer. Here, we describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis of methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes. DREAM provides information on 150,000 unique CpG sites, of which 39,000 are in CpG islands and 30,000 are at transcription start sites of 13,000 RefSeq genes. We analyzed DNA methylation in healthy white blood cells and found methylation patterns to be remarkably uniform. Inter individual differences > 30% were observed only at 227 of 28,331 (0.8%) of autosomal CpG sites. Similarly, > 30% differences were observed at only 59 sites when we comparing the cord and adult blood. These conserved methylation patterns contrasted with extensive changes affecting 18-40% of CpG sites in a patient with acute myeloid leukemia and in two leukemia cell lines. The method is cost effective, quantitative (r ( 2) = 0.93 when compared with bisulfite pyrosequencing) and reproducible (r ( 2) = 0.997). Using 100-fold coverage, DREAM can detect differences in methylation greater than 10% or 30% with a false positive rate below 0.05 or 0.001, respectively. DREAM can be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes.

  16. Quantitative and correlation analysis of the DNA methylation and expression of DAPK in breast cancer

    Science.gov (United States)

    Zhu, Youzhi; Li, Shuiqin; Wang, Qingshui; Chen, Ling; Wu, Kunlin; Huang, Yide

    2017-01-01

    Background Death-associated protein kinase 1 (DAPK) is an important tumor suppressor kinase involved in the regulation of multiple cellular activities such as apoptosis and autophagy. DNA methylation of DAPK gene was found in various types of cancers and often correlated with the clinicopathological characteristics. However, the mRNA and protein expression of DAPK in the same sample was rarely measured. Thus, it was unclear if the correlation between DAPK gene methylation and clinicopathological parameters was due to the loss of DAPK expression. Methods In this study, the DNA methylation rate, mRNA and protein expression of DAPK was quantitatively detected in 15 pairs of breast cancer patient samples including tumor (T) and adjacent non-tumor (N) tissues. Results The correlation between DNA methylation rate and mRNA expression, together with the correlation between mRNA and protein expression, was calculated. No correlation was observed between any levels using either the measurement value of each sample or the T/N ratio of each pair. Discussion These data suggested that the DNA methylation status of DAPK did not correlate well with its mRNA or protein expression. Extra caution is needed when interpreting the DNA methylation data of DAPK gene in clinical studies.

  17. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

    Institute of Scientific and Technical Information of China (English)

    XU Chun-hai; LI Zhao-shen; DAI Jun-ying; ZHU Hai-yang; YU Jian-wu; L(U) Shu-Ian

    2011-01-01

    peripheral blood mononuclear cells; marrow mononuclear cells Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0x102 copies/ml to 3.9x108 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

  18. Quantitative PCR - new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situhybridization

    DEFF Research Database (Denmark)

    Schlemmer, B.O.; Sørensen, B. S.; Overgaard, J.

    2004-01-01

    Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression......, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...

  19. Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

    Science.gov (United States)

    Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

    2014-02-01

    DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

  20. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    Science.gov (United States)

    Singh, Vijay Raj; So, Peter T. C.

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

  1. Changes in the biological diversity and concentration of total DNA under the influence of mineral fertilizers in agrochernozemic soils

    Science.gov (United States)

    Tkhakakhova, Azida; Kutovaya, Olga; Ivanova, Ekaterina; Pavlyuchenko, Anatoly

    2014-05-01

    Chernozems represent the most valuable soil resource for Russian agriculture. Their sustainable use in intensive farming systems with preservation of the biological diversity and biological activity of these soils is of crucial importance for the agri-environmental security of Russia. We studied the influence of different rates of mineral fertilizers on the biological activity of chernozems on experimental fields of the Dokuchaev Research Institute of Agriculture in Kamennaya Steppe (Voronezh oblast). Soil samples were taken at the end of April 2013 from the plow horizon on trials with different rates of fertilization: NPK-0, NPK-60, and NPK-120 (kg/ha); a long-term fallow plot was used as an absolute control. The biological activity was analyzed by routine inoculation methods and by the molecular biology techniques based on DNA isolation from the soil samples. Quantitative parameters of the isolated and purified DNA were determined by measuring the fluorescence of the DNA preparations with added intercalating dyes; GelDoc XR system and Image Lab and TotalLab Quant. software were used. Microbiological studies showed the high biological activity of the chernozems soil in all the trials. No significant differences were found between the trials for the microbiological processes of the carbon cycle. There was a weakly expressed tendency for an increase in the activity of actinomycetes from the soil with zero fertilization (5.11 log10CFU/g) to the soil with maximum (NPK-120) fertilization (5.69 log10CFU/g) and the fallow soil (5.73 log10CFU/g); the number of cultivated micromycetes decreased from the soil with zero fertilization (4.76 log10CFU/g) to the soil with maximum fertilization (4.14 log10CFU/g) and to the fallow soil (4.1 log10CFU/g). A less equilibrium state is typical of the microorganisms participating in the nitrogen cycle. The number of cultivated aerobic and anaerobic nitrogen-fixing bacteria somewhat increased in the fertilized trials (NPK-60, NPK-120

  2. Evidence for DNA Damage as a Biological Link Between Diabetes and Cancer

    Institute of Scientific and Technical Information of China (English)

    Shao Chin Lee; Juliana CN Chan

    2015-01-01

    Objective:This review examines the evidence that:Diabetes is a state of DNA damage;pathophysiological factors in diabetes can cause DNA damage;DNA damage can cause mutations;and DNA mutation is linked to carcinogenesis.Data Sources:We retrieved information from the PubMed database up to January,2014,using various search terms and their combinations including DNA damage,diabetes,cancer,high glucose,hyperglycemia,free fatty acids,palmitic acid,advanced glycation end products,mutation and carcinogenesis.Study Selection:We included data from peer-reviewed journals and a textbook printed in English on relationships between DNA damage and diabetes as well as pathophysiological factors in diabetes.Publications on relationships among DNA damage,mutagenesis,and carcinogenesis,were also reviewed.We organized this information into a conceptual framework to explain the possible causal relationship between DNA damage and carcinogenesis in diabetes.Results:There are a large amount of data supporting the view that DNA mutation is a typical feature in carcinogenesis.Patients with type 2 diabetes have increased production of reactive oxygen species,reduced levels of antioxidant capacity,and increased levels of DNA damage.The pathophysiological factors and metabolic milieu in diabetes can cause DNA damage such as DNA strand break and base modification (i.e.,oxidation).Emerging experimental data suggest that signal pathways (i.e.,Akt/tuberin) link diabetes to DNA damage.This collective evidence indicates that diabetes is a pathophysiological state of oxidative stress and DNA damage which can lead to various types of mutation to cause aberration in cells and thereby increased cancer risk.Conclusions:This review highlights the interrelationships amongst diabetes,DNA damage,DNA mutation and carcinogenesis,which suggests that DNA damage can be a biological link between diabetes and cancer.

  3. Quantitative and dynamic measurements of biological fresh samples with X-ray phase contrast tomography

    Energy Technology Data Exchange (ETDEWEB)

    Hoshino, Masato, E-mail: hoshino@spring8.or.jp; Uesugi, Kentaro [Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan); Tsukube, Takuro [Japanese Red Cross Kobe Hospital, 1-3-1 Wakinohamakaigandori, Chuo-ku, Kobe, Hyogo 651-0073 (Japan); Yagi, Naoto [Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2014-10-08

    Quantitative measurements of biological fresh samples based on three-dimensional densitometry using X-ray phase contrast tomography are presented. X-ray phase contrast tomography using a Talbot grating interferometer was applied to biological fresh samples which were not fixed by any fixatives. To achieve a high-throughput measurement for the fresh samples the X-ray phase contrast tomography measurement procedure was improved. The three-dimensional structure of a fresh mouse fetus was clearly depicted as a mass density map using X-ray phase contrast tomography. The mouse fetus measured in the fresh state was then fixed by formalin and measured in the fixed state. The influence of the formalin fixation on soft tissue was quantitatively evaluated by comparing the fresh and fixed samples. X-ray phase contrast tomography was also applied to the dynamic measurement of a biological fresh sample. Morphological changes of a ring-shaped fresh pig aorta were measured tomographically under different degrees of stretching.

  4. Is there any measurable benefit in publishing preprints in the arXiv section Quantitative Biology?

    CERN Document Server

    Aman, Valeria

    2014-01-01

    A public preprint server such as arXiv allows authors to publish their manuscripts before submitting them to journals for peer review. It offers the chance to establish priority by making the results available upon completion. This article presents the arXiv section Quantitative Biology and investigates the advantages of preprint publications in terms of reception, which can be measured by means of citations. This paper focuses on the publication and citation delay, citation counts and the authors publishing their e-prints on arXiv. Moreover, the paper discusses the benefit for scientists as well as publishers. The results that are based on 12 selected journals show that submitting preprints to arXiv has become more common in the past few years, but the number of papers submitted to Quantitative Biology is still small and represents only a fraction of the total research output in biology. An immense advantage of arXiv is to overcome the long publication delay resulting from peer review. Although preprints are...

  5. DNA confinement in nanochannels: physics and biological applications

    DEFF Research Database (Denmark)

    Reisner, Walter; Pedersen, Jonas Nyvold; Austin, Robert H

    2012-01-01

    in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense...

  6. Quantitative model analysis with diverse biological data: applications in developmental pattern formation.

    Science.gov (United States)

    Pargett, Michael; Umulis, David M

    2013-07-15

    Mathematical modeling of transcription factor and signaling networks is widely used to understand if and how a mechanism works, and to infer regulatory interactions that produce a model consistent with the observed data. Both of these approaches to modeling are informed by experimental data, however, much of the data available or even acquirable are not quantitative. Data that is not strictly quantitative cannot be used by classical, quantitative, model-based analyses that measure a difference between the measured observation and the model prediction for that observation. To bridge the model-to-data gap, a variety of techniques have been developed to measure model "fitness" and provide numerical values that can subsequently be used in model optimization or model inference studies. Here, we discuss a selection of traditional and novel techniques to transform data of varied quality and enable quantitative comparison with mathematical models. This review is intended to both inform the use of these model analysis methods, focused on parameter estimation, and to help guide the choice of method to use for a given study based on the type of data available. Applying techniques such as normalization or optimal scaling may significantly improve the utility of current biological data in model-based study and allow greater integration between disparate types of data. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Application of LC-MS/MS for quantitative analysis of glucocorticoids and stimulants in biological fluids

    Institute of Scientific and Technical Information of China (English)

    Jamshed Haneef; Mohammad Shaharyar; Asif Husaina; Mohd Rashid; Ravinesh Mishra; Shama Parveen; Niyaz Ahmed; Manoj Pal; Deepak Kumar

    2013-01-01

    Liquid chromatography tandem mass chromatography (LC-MS/MS) is an important hyphenated technique for quantitative analysis of drugs in biological fluids. Because of high sensitivity and selectivity, LC-MS/MS has been used for pharmacokinetic studies, metabolites identification in the plasma and urine. This manuscript gives comprehensive analytical review, focusing on chromatographic separation approaches (column packing materials, column length and mobile phase) as well as different acquisition modes (SIM, MRM) for quantitative analysis of glucocorticoids and stimulants. This review is not meant to be exhaustive but rather to provide a general overview for detection and confirmation of target drugs using LC-MS/MS and thus useful in the doping analysis, toxicological studies as well as in pharmaceutical analysis.

  8. Evaluating the weight of evidence by using quantitative short tandem repeat data in DNA mixtures

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2010-01-01

    he evaluation of results from mixtures of deoxyribonucleic acid (DNA) from two or more people in crime case investigations may be improved by taking not only the qualitative but also the quantitative part of the results into consideration. We present a statistical likelihood approach to assess...... distribution of peak areas for assessing the weight of the evidence. On the basis of data from analyses of controlled experiments with mixed DNA samples, we exploited the linear relationship between peak heights and peak areas, and the linear relationships of the means and variances of the measurements...... to factorization of the likelihood, properties of the normal distribution and use of auxiliary variables, an ordinary implementation of the EM algorithm solved the missing data problem....

  9. Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    YANG Zong-zhao; HABIB Mudasser; SHUAI Jiang-bing; FANG Wei-huan

    2007-01-01

    We developed an assay for the detection and quantitation ofporcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction.No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

  10. Dual mode diffraction phase microscopy for quantitative functional assessment of biological cells

    Science.gov (United States)

    Talaikova, N. A.; Popov, A. P.; Kalyanov, A. L.; Ryabukho, V. P.; Meglinski, I. V.

    2017-10-01

    A diffraction phase microscopy approach with a combined use of transmission and reflection imaging modes has been developed and applied for non-invasive quantitative assessment of the refractive index of red blood cells (RBCs). We present the theoretical background of signal formation for both imaging modes, accompanied by the results of experimental studies. We demonstrate that simultaneous use of the two modes has great potential for accurate assessment of the refractive index of biological cells, and we perform a reconstruction of spatial distribution of the refractive index of RBC in 3D.

  11. Quantitative Analysis of Clustered DNA Damages Induced by Silicon Beams of Different Kinetic Energy

    Energy Technology Data Exchange (ETDEWEB)

    Keszenman D. J.; Keszenman, D.J.; Bennett, P.V.; Sutherland, B.M.; Wilson, P.F.

    2013-05-14

    Humans may b exposed to highly energetic charged particle radiation as a result of medical treatments, occupational activitie or accidental events. In recent years, our increasing presence and burgeoning interest in space exploration beyond low Earth orbit has led to a large increase in the research of the biological effects ofcharged particle radiation typical of that encountered in the space radiation environment. The study of the effects of these types of radiation qualities in terms ofDNA damage induction and repair is fundamental to understand mechanisms both underlying their greater biological effectiveness as we)) as the short and long term risks of health effects such as carcinogenesis, degen rative diseases and premature aging. Charged particle radiation induces a variety of DNA alterations, notably bistranded clustered damages, defined as two or more closely-opposed strand break , oxidized bases or abasic sites within a few helical turns. The induction of such highly complex DNA damage enhances the probability of incorrect or incomplete repair and thus constitutes greater potential for genomic instability, cell death and transformation.

  12. Introduction to the Symposium "Leading Students and Faculty to Quantitative Biology through Active Learning".

    Science.gov (United States)

    Waldrop, Lindsay D; Miller, Laura A

    2015-11-01

    The broad aim of this symposium and set of associated papers is to motivate the use of inquiry-based, active-learning teaching techniques in undergraduate quantitative biology courses. Practical information, resources, and ready-to-use classroom exercises relevant to physicists, mathematicians, biologists, and engineers are presented. These resources can be used to address the lack of preparation of college students in STEM fields entering the workforce by providing experience working on interdisciplinary and multidisciplinary problems in mathematical biology in a group setting. Such approaches can also indirectly help attract and retain under-represented students who benefit the most from "non-traditional" learning styles and strategies, including inquiry-based, collaborative, and active learning.

  13. Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry.

    Science.gov (United States)

    Hopfgartner, Gérard; Bourgogne, Emmanuel

    2003-01-01

    To support pharmacokinetic and drug metabolism studies, LC-MS/MS plays more and more an essential role for the quantitation of drugs and their metabolites in biological matrices. With the new challenges encountered in drug discovery and drug development, new strategies are put in place to achieve high-throughput analysis, using serial and parallel approaches. To speed-up method development and validation, generic approaches with the direct injection of biological fluids is highly desirable. Column-switching, using various packing materials for the extraction columns, is widely applied. Improvement of mass spectrometers performance, and in particular triple quadrupoles, also strongly influences sample preparation strategies, which remain a key element in the bioanalytical process. Copyright 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:195-214, 2003; Published online in Wiley Interscience (www.interscience.wiley.com). DOI 10.1002/mas.10050

  14. Biological Sensors Using DNA Functionalized Multiwalled Carbon Nanotubes

    Science.gov (United States)

    2009-10-01

    out using potassium ferro cyanide as the redox electrolyte. Cyclic voltammetry gave well-defined quasi-reversible voltammetric responses, flat and...performance studies have been carried out using potassium ferro cyanide as the redox electrolyte and DNA-MWNT film electrode and results have been discussed...for the synthesis of MWNT in large quantities using Mischmetal (Bharat Rare Earths Metals, India; composition:- Ce 50%, La 35%, Pr 8 %, Nd 5%, Fe

  15. Synthesis and biological activity of benzamide DNA minor groove binders.

    Science.gov (United States)

    Khan, Gul Shahzada; Pilkington, Lisa I; Barker, David

    2016-02-01

    A range of di- and triaryl benzamides were synthesised to investigate the effect of the presence and nature of a polar sidechain, bonding and substitution patterns and functionalisation of benzylic substituents. These compounds were tested for their antiproliferative activity as well as their DNA binding activity. The most active compounds in all assays were unsymmetrical triaryl benzamides with a bulky or alkylating benzylic substituent and a polar amino sidechain.

  16. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    Science.gov (United States)

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns.

  17. AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples.

    Science.gov (United States)

    Liu, Jason Y; Zhong, Chang; Holt, Allison; Lagace, Robert; Harrold, Michael; Dixon, Alan B; Brevnov, Maxim G; Shewale, Jaiprakash G; Hennessy, Lori K

    2012-07-01

    The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

  18. Quantitative computational models of molecular self-assembly in systems biology

    Science.gov (United States)

    Thomas, Marcus; Schwartz, Russell

    2017-06-01

    Molecular self-assembly is the dominant form of chemical reaction in living systems, yet efforts at systems biology modeling are only beginning to appreciate the need for and challenges to accurate quantitative modeling of self-assembly. Self-assembly reactions are essential to nearly every important process in cell and molecular biology and handling them is thus a necessary step in building comprehensive models of complex cellular systems. They present exceptional challenges, however, to standard methods for simulating complex systems. While the general systems biology world is just beginning to deal with these challenges, there is an extensive literature dealing with them for more specialized self-assembly modeling. This review will examine the challenges of self-assembly modeling, nascent efforts to deal with these challenges in the systems modeling community, and some of the solutions offered in prior work on self-assembly specifically. The review concludes with some consideration of the likely role of self-assembly in the future of complex biological system models more generally.

  19. Life at the Common Denominator: Mechanistic and Quantitative Biology for the Earth and Space Sciences

    Science.gov (United States)

    Hoehler, Tori M.

    2010-01-01

    The remarkable challenges and possibilities of the coming few decades will compel the biogeochemical and astrobiological sciences to characterize the interactions between biology and its environment in a fundamental, mechanistic, and quantitative fashion. The clear need for integrative and scalable biology-environment models is exemplified in the Earth sciences by the challenge of effectively addressing anthropogenic global change, and in the space sciences by the challenge of mounting a well-constrained yet sufficiently adaptive and inclusive search for life beyond Earth. Our understanding of the life-planet interaction is still, however, largely empirical. A variety of approaches seek to move from empirical to mechanistic descriptions. One approach focuses on the relationship between biology and energy, which is at once universal (all life requires energy), unique (life manages energy flow in a fashion not seen in abiotic systems), and amenable to characterization and quantification in thermodynamic terms. Simultaneously, a focus on energy flow addresses a critical point of interface between life and its geological, chemical, and physical environment. Characterizing and quantifying this relationship for life on Earth will support the development of integrative and predictive models for biology-environment dynamics. Understanding this relationship at its most fundamental level holds potential for developing concepts of habitability and biosignatures that can optimize astrobiological exploration strategies and are extensible to all life.

  20. Recursively partitioned mixture model clustering of DNA methylation data using biologically informed correlation structures.

    Science.gov (United States)

    Koestler, Devin C; Christensen, Brock C; Marsit, Carmen J; Kelsey, Karl T; Houseman, E Andres

    2013-03-05

    DNA methylation is a well-recognized epigenetic mechanism that has been the subject of a growing body of literature typically focused on the identification and study of profiles of DNA methylation and their association with human diseases and exposures. In recent years, a number of unsupervised clustering algorithms, both parametric and non-parametric, have been proposed for clustering large-scale DNA methylation data. However, most of these approaches do not incorporate known biological relationships of measured features, and in some cases, rely on unrealistic assumptions regarding the nature of DNA methylation. Here, we propose a modified version of a recursively partitioned mixture model (RPMM) that integrates information related to the proximity of CpG loci within the genome to inform correlation structures from which subsequent clustering analysis is based. Using simulations and four methylation data sets, we demonstrate that integrating biologically informative correlation structures within RPMM resulted in improved goodness-of-fit, clustering consistency, and the ability to detect biologically meaningful clusters compared to methods which ignore such correlation. Integrating biologically-informed correlation structures to enhance modeling techniques is motivated by the rapid increase in resolution of DNA methylation microarrays and the increasing understanding of the biology of this epigenetic mechanism.

  1. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

  2. A quantitative model of human DNA base excision repair. I. Mechanistic insights.

    Science.gov (United States)

    Sokhansanj, Bahrad A; Rodrigue, Garry R; Fitch, J Patrick; Wilson, David M

    2002-04-15

    Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polbeta-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polbeta-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity.

  3. Integrating ethical analysis "into the DNA" of synthetic biology.

    Science.gov (United States)

    Heavey, Patrick

    2015-02-01

    Current ethical analysis tends to evaluate synthetic biology at an overview level. Synthetic biology, however, is an umbrella term that covers a variety of areas of research. These areas contain, in turn, a hierarchy of different research fields. This abstraction hierarchy-the term is borrowed from engineering-permits synthetic biologists to specialise to a very high degree. Though synthetic biology per se may create profound ethical challenges, much of the day-to-day research does not. Yet seemingly innocuous research could lead to ethically problematic results. For example, Dolly the sheep resulted from a long series of research steps, none of which presented any ethical problems. The atomic bomb was developed as a result of Einstein's uncontentions theoretical research that proved the equivalence of matter and energy. Therefore it would seem wise for ethicists to evaluate synbio research across its subfields and through its abstraction hierarchies, comparing and inter-relating the various areas of research. In addition, it would be useful if journals that publish synbio papers require an ethical statement from authors, as standard practice, so as to encourage scientists to constantly engage with ethical issues in their work. Also, this would allow an ethical snapshot of the state of the research at any given time to exist, allowing for accurate evaluation by scientists and ethicists, regulators and policymakers.

  4. Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Straube Eberhard

    2010-04-01

    Full Text Available Abstract Background The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Methods Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN, peqGold™ Tissue DNA Mini Kit (PeqLab, UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio, DNA Isolation Kit for Cells and Tissues (Roche, and NucleoSpin™ Tissue (Macherey-Nagel. DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. Results Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p Conclusions We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

  5. Quantitative measurement of porphyrins in biological tissues and evaluation of tissue porphyrins during toxicant exposures.

    Science.gov (United States)

    Woods, J S; Miller, H D

    1993-10-01

    Porphyrins are formed in most eukaryotic tissues as intermediates in the biosynthesis of heme. Assessment of changes in tissue porphyrin levels occurring in response to the actions of various drugs or toxicants is potentially useful in the evaluation of chemical exposures and effects. The present paper describes a rapid and sensitive method for the extraction and quantitation of porphyrins in biological tissues which overcomes difficulties encountered in previously described methods, particularly the loss of porphyrins during extraction and interference of porphyrin quantitation by coeluting fluorescent tissue constituents. In this procedure 8- through 2-carboxyl porphyrins are quantitatively extracted from tissue homogenates using HCl and methanol and are subsequently separated from potentially interfering contaminants by sequential methanol/phosphate elution on a C-18 preparatory column. Porphyrins are then separated and measured by reversed-phase high-performance liquid chromatography and spectrofluorometric techniques. Recovery of tissue porphyrins using this method is close to 100% with an intraassay variability of less than 10%. We have employed this procedure to measure liver and kidney porphyrin concentrations in male Fischer rats and to define the distinctive changes in tissue porphyrin patterns associated with treatment with the hepatic and renal porphyrinogenic chemicals, allylisopropylacetamide, and methyl mercury hydroxide, respectively. This method is applicable to the measurement of tissue porphyrin changes resulting from drug or toxicant exposures in clinical, experimental or environmental assessments.

  6. Closing the Loop: Involving Faculty in the Assessment of Scientific and Quantitative Reasoning Skills of Biology Majors

    Science.gov (United States)

    Hurney, Carol A.; Brown, Justin; Griscom, Heather Peckham; Kancler, Erika; Wigtil, Clifton J.; Sundre, Donna

    2011-01-01

    The development of scientific and quantitative reasoning skills in undergraduates majoring in science, technology, engineering, and mathematics (STEM) is an objective of many courses and curricula. The Biology Department at James Madison University (JMU) assesses these essential skills in graduating biology majors by using a multiple-choice exam…

  7. Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

    Science.gov (United States)

    Benassi, Julia Cristina; Benvenga, Graziella U; Ferreira, Helena Lage; Pereira, Vanessa F; Keid, Lara B; Soares, Rodrigo; Oliveira, Tricia Maria Ferreira de Sousa

    2017-06-01

    Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

    Science.gov (United States)

    Tretyakova, Natalia Y; Groehler, Arnold; Ji, Shaofei

    2015-06-16

    Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural

  9. A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

    DEFF Research Database (Denmark)

    Blasius, Melanie; Wagner, Sebastian A; Choudhary, Chuna Ram

    2014-01-01

    RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome...

  10. Quantitative bioluminometric method for DNA-based species/varietal identification in food authenticity assessment.

    Science.gov (United States)

    Trantakis, Ioannis A; Christopoulos, Theodore K; Spaniolas, Stelios; Kalaitzis, Panagiotis; Ioannou, Penelope C; Tucker, Gregory A

    2012-02-01

    A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca(2+). Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.

  11. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

    Science.gov (United States)

    Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

  12. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp..

    Directory of Open Access Journals (Sweden)

    Cameron R Turner

    Full Text Available Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp., an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

  13. Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

    Science.gov (United States)

    Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

  14. Chemistry and Biology of Aflatoxin-DNA Adducts

    Energy Technology Data Exchange (ETDEWEB)

    Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.; Egli, Martin (Vanderbilt)

    2012-03-27

    Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate above the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.

  15. 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...... to the synthetic biology community....

  16. DNA extraction protocol for biological ingredient analysis of Liuwei Dihuang Wan.

    Science.gov (United States)

    Cheng, Xinwei; Chen, Xiaohua; Su, Xiaoquan; Zhao, Huanxin; Han, Maozhen; Bo, Cunpei; Xu, Jian; Bai, Hong; Ning, Kang

    2014-06-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations. Copyright © 2014. Production and hosting by Elsevier Ltd.

  17. DNA Extraction Protocol for Biological Ingredient Analysis of Liuwei Dihuang Wan

    Institute of Scientific and Technical Information of China (English)

    Xinwei Cheng; Xiaohua Chen; Xiaoquan Su; Huanxin Zhao; Maozhen Han; Cunpei Bo; Jian Xu; Hong Bai; Kang Ning

    2014-01-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological con-taminants. For biological ingredient analysis, the efficiency of DNA extraction is an important fac-tor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three com-monly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out.The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations.

  18. Electrochemical biosensor for quantitation of anti-DNA autoantibodies in human serum.

    Science.gov (United States)

    Rubin, Robert L; Wall, David; Konstantinov, Konstantin N

    2014-01-15

    Measurement of serum autoantibody is a critical tool in the diagnosis and management of autoimmune diseases. However, rapid and convenient methods at the point-of care have not been achieved in large part because any one antibody species is a heterogeneous and miniscule fraction of the total serum immunoglobulin displaying identical properties other than its antigen-binding specificity. The present system addresses these challenges by vacuum-mediated transport of diluted serum through an antigen-coated porous membrane. To measure anti-DNA autoantibodies, native DNA was immobilized into a poly(vinylidene fluoride) membrane pre-coated with a synthetic phenylalanine/lysine co-polymer. Flow-through of primary and peroxidase-conjugated secondary antibodies over the course of 3 min enhanced productive antibody-antigen interactions by bringing the reactants into close mutual proximity. Signal was quantified electrochemically during the enzymatic conversion of the tetramethylbenzidine substrate to a charge-transfer complex. The electrochemical signals generated by sera from patients with systemic lupus erythematosus using this device showed good quantitative correlation with a standard enzyme-linked immunosorbent assay and displayed similar detection limits. Inter- and intra-assay variability and electrode uniformity were favorable as was a two-month test of the stability of the DNA-coated membrane. While refining the fluidics requirements of this biosensor will be needed, its capacity to quantify over the course of 30 min anti-DNA antibodies in fresh human serum without background reactivity of normal serum makes this a promising technology as a point-of care device of clinical utility.

  19. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA

    Science.gov (United States)

    Hunter, Margaret; Dorazio, Robert M.; Butterfield, John S.; Meigs-Friend, Gaia; Nico, Leo; Ferrante, Jason

    2017-01-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics.

  20. A DNA-Based Encryption Method Based on Two Biological Axioms of DNA Chip and Polymerase Chain Reaction (PCR) Amplification Techniques.

    Science.gov (United States)

    Zhang, Yunpeng; Wang, Zhiwen; Wang, Zhenzhen; Liu, Xin; Yuan, Xiaojing

    2017-09-27

    Researchers have gained a deeper understanding of DNA-based encryption and its effectiveness in enhancing information security in recent years. However, there are many theoretical and technical issues about DNA-based encryption that need to be addressed before it can be effectively used in the field of security. Currently, the most popular DNA-based encryption schemes are based on traditional cryptography and the integration of existing DNA technology. These schemes are not completely based on DNA computing and biotechnology. Herein, as inspired by nature, encryption based on DNA has been developed, which is, in turn, based on two fundamental biological axioms about DNA sequencing: 1) DNA sequencing is difficult under the conditions of not knowing the correct sequencing primers and probes, and 2) without knowing the correct probe, it is difficult to decipher precisely and sequence the information of unknown and mixed DNA/peptide nucleic acid (PNA) probes, which only differ in nucleotide sequence, arranged on DNA chips (microarrays). In essence, when creating DNA-based encryption by means of biological technologies, such as DNA chips and polymerase chain reaction (PCR) amplification, the encryption method discussed herein cannot be decrypted, unless the DNA/PNA probe or PCR amplification is known. The biological analysis, mathematical analysis, and simulation results demonstrate the feasibility of the method, which provides much stronger security and reliability than that of traditional encryption methods. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

    Science.gov (United States)

    Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

    2013-10-01

    A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry.

  2. Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples.

    Science.gov (United States)

    Morales, J M; Jung, C H; Alarcón, A; Barreda, A

    2000-09-15

    This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 microm) using water-acetonitrile-methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 microg/ml. The limit of quantitation was 0.08 microg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.

  3. Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems.

    Science.gov (United States)

    Mesquita, Daniela P; Leal, Cristiano; Cunha, Jorge R; Oehmen, Adrian; Amaral, A Luís; Reis, Maria A M; Ferreira, Eugénio C

    2013-04-03

    The present study focuses on predicting the concentration of intracellular storage polymers in enhanced biological phosphorus removal (EBPR) systems. For that purpose, quantitative image analysis techniques were developed for determining the intracellular concentrations of PHA (PHB and PHV) with Nile blue and glycogen with aniline blue staining. Partial least squares (PLS) were used to predict the standard analytical values of these polymers by the proposed methodology. Identification of the aerobic and anaerobic stages proved to be crucial for improving the assessment of PHA, PHB and PHV intracellular concentrations. Current Nile blue based methodology can be seen as a feasible starting point for further enhancement. Glycogen detection based on the developed aniline blue staining methodology combined with the image analysis data proved to be a promising technique, toward the elimination of the need for analytical off-line measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.

    Directory of Open Access Journals (Sweden)

    Yang An

    Full Text Available A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.

  5. Quantitative utilization of prior biological knowledge in the Bayesian network modeling of gene expression data

    Directory of Open Access Journals (Sweden)

    Gao Shouguo

    2011-08-01

    Full Text Available Abstract Background Bayesian Network (BN is a powerful approach to reconstructing genetic regulatory networks from gene expression data. However, expression data by itself suffers from high noise and lack of power. Incorporating prior biological knowledge can improve the performance. As each type of prior knowledge on its own may be incomplete or limited by quality issues, integrating multiple sources of prior knowledge to utilize their consensus is desirable. Results We introduce a new method to incorporate the quantitative information from multiple sources of prior knowledge. It first uses the Naïve Bayesian classifier to assess the likelihood of functional linkage between gene pairs based on prior knowledge. In this study we included cocitation in PubMed and schematic similarity in Gene Ontology annotation. A candidate network edge reservoir is then created in which the copy number of each edge is proportional to the estimated likelihood of linkage between the two corresponding genes. In network simulation the Markov Chain Monte Carlo sampling algorithm is adopted, and samples from this reservoir at each iteration to generate new candidate networks. We evaluated the new algorithm using both simulated and real gene expression data including that from a yeast cell cycle and a mouse pancreas development/growth study. Incorporating prior knowledge led to a ~2 fold increase in the number of known transcription regulations recovered, without significant change in false positive rate. In contrast, without the prior knowledge BN modeling is not always better than a random selection, demonstrating the necessity in network modeling to supplement the gene expression data with additional information. Conclusion our new development provides a statistical means to utilize the quantitative information in prior biological knowledge in the BN modeling of gene expression data, which significantly improves the performance.

  6. DNA methylation-based measures of biological age: Meta-analysis predicting time to death

    NARCIS (Netherlands)

    B.H. Chen (Brian); R.E. Marioni (Riccardo); Colicino, E. (Elena); M.J. Peters (Marjolein); C.K. Ward-Caviness (Cavin K.); Tsai, P.-C. (Pei-Chien); Roetker, N.S. (Nicholas S.); Just, A.C. (Allan C.); E.W. Demerath (Ellen); W. Guan (Weihua); J. Bressler (Jan); M. Fornage (Myriam); Studenski, S. (Stephanie); Vandiver, A.R. (Amy R.); Moore, A.Z. (Ann Zenobia); T. Tanaka (Toshiko); D.P. Kiel (Douglas P.); Liang, L. (Liming); Vokonas, P. (Pantel); Schwartz, J. (Joel); K.L. Lunetta (Kathryn); J. Murabito (Joanne); S. Bandinelli (Stefania); D.G. Hernandez (Dena); D. Melzer (David); M.A. Nalls (Michael); L.C. Pilling (Luke); Price, T.R. (Timothy R.); A. Singleton (Andrew); C. Gieger (Christian); R. Holle (Rolf); Kretschmer, A. (Anja); F. Kronenberg (Florian); Kunze, S. (Sonja); J. Linseisen (Jakob); Meisinger, C. (Christine); W. Rathmann (Wolfgang); M. Waldenberger (Melanie); P.M. Visscher (Peter); Shah, S. (Sonia); N.R. Wray (Naomi); A.F. McRae (Allan F.); O.H. Franco (Oscar); A. Hofman (Albert); A.G. Uitterlinden (André); D. Absher (Devin); T.L. Assimes (Themistocles); Levine, M.E. (Morgan E.); Lu, A.T. (Ake T.); Tsao, P.S. (Philip S.); Hou, L. (Lifang); J.E. Manson (Joann); C. Carty (Cara); LaCroix, A.Z. (Andrea Z.); A. Reiner (Alexander); T.D. Spector (Timothy); A.P. Feinberg (Andrew P.); D. Levy (Daniel); A.A. Baccarelli (Andrea A.); Meurs, J. (Joyce van); J.T. Bell (Jordana); A. Peters (Annette); I.J. Deary (Ian J.); J.S. Pankow (James); L. Ferrucci (Luigi); S. Horvath (Steve)

    2016-01-01

    textabstractEstimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cau

  7. Capacity for DNA-barcode based taxonomy in support of Great Lakes biological monitoring

    Science.gov (United States)

    Enumerating organisms collected via nets and sediment grabs is a mainstay of aquatic ecology. Since morphological taxonomy can require considerable resources and expertise, DNA barcode-based identification of mixed-organism samples offers a valuable tool in support of biological...

  8. Microwave-accelerated bioassay technique for rapid and quantitative detection of biological and environmental samples.

    Science.gov (United States)

    Mohammed, Muzaffer; Syed, Maleeha F; Aslan, Kadir

    2016-01-15

    Quantitative detection of molecules of interest from biological and environmental samples in a rapid manner, particularly with a relevant concentration range, is imperative to the timely assessment of human diseases and environmental issues. In this work, we employed the microwave-accelerated bioassay (MAB) technique, which is based on the combined use of circular bioassay platforms and microwave heating, for rapid and quantitative detection of Glial Fibrillary Acidic Protein (GFAP) and Shiga like toxin (STX 1). The proof-of-principle use of the MAB technique with the circular bioassay platforms for the rapid detection of GFAP in buffer based on colorimetric and fluorescence readouts was demonstrated with a 900W kitchen microwave. We also employed the MAB technique with a new microwave system (called the iCrystal system) for the detection of GFAP from mice with brain injuries and STX 1 from a city water stream. Control bioassays included the commercially available gold standard bioassay kits run at room temperature. Our results show that the lower limit of detection (LLOD) of the colorimetric and fluorescence based bioassays for GFAP was decreased by ~1000 times using the MAB technique and our circular bioassay platforms as compared to the commercially available bioassay kits. The overall bioassay time for GFAP and STX 1 was reduced from 4h using commercially available bioassay kits to 10min using the MAB technique.

  9. Enantioselective reductive transformation of climbazole: A concept towards quantitative biodegradation assessment in anaerobic biological treatment processes.

    Science.gov (United States)

    Brienza, Monica; Chiron, Serge

    2017-06-01

    An efficient chiral method-based using liquid chromatography-high resolution-mass spectrometry analytical method has been validated for the determination of climbazole (CBZ) enantiomers in wastewater and sludge with quantification limits below the 1 ng/L and 2 ng/g range, respectively. On the basis of this newly developed analytical method, the stereochemistry of CBZ was investigated over time in sludge biotic and sterile batch experiments under anoxic dark and light conditions and during wastewater biological treatment by subsurface flow constructed wetlands. CBZ stereoselective degradation was exclusively observed under biotic conditions, confirming the specificity of enantiomeric fraction variations to biodegradation processes. Abiotic CBZ enantiomerization was insignificant at circumneutral pH and CBZ was always biotransformed into CBZ-alcohol due to the specific and enantioselective reduction of the ketone function of CBZ into a secondary alcohol function. This transformation was almost quantitative and biodegradation gave good first order kinetic fit for both enantiomers. The possibility to apply the Rayleigh equation to enantioselective CBZ biodegradation processes was investigated. The results of enantiomeric enrichment allowed for a quantitative assessment of in situ biodegradation processes due to a good fit (R(2) > 0.96) of the anoxic/anaerobic CBZ biodegradation to the Rayleigh dependency in all the biotic microcosms and was also applied in subsurface flow constructed wetlands. This work extended the concept of applying the Rayleigh equation towards quantitative biodegradation assessment of organic contaminants to enantioselective processes operating under anoxic/anaerobic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    Science.gov (United States)

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. BORRELIA BURGDORFERI DNA IN BIOLOGICAL SAMPLES FROM PATIENTS WITH SARCOIDOSIS USING THE POLYMERASE CHAIN REACTION TECHNIQUE

    Institute of Scientific and Technical Information of China (English)

    连伟; 罗慰慈

    1995-01-01

    Polymerase chain reaction (PCR) was used to detect the presence of Borretia burgdoferi DNA in biological samples from patients with sarcoidcsis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridlzation with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdor feri genome, even in the presence of a 104-fold excess of human eukaryotic DNA, and was also specific to different B. burgdorferl strains tested. Sera seroiogieally positive to B. burgdorferi (n=26), broncbemlveolar lavage fluid and supematant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26, and 0/9, respectively). It was considered that DNA from B. bur gdor feri may be identified in a minority of patients with s,arcoidosis, and it may play a pathogenetic rote in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.

  12. Coherent optical spectroscopy in a biological semiconductor quantum dot-DNA hybrid system

    Science.gov (United States)

    2012-01-01

    We theoretically investigate coherent optical spectroscopy of a biological semiconductor quantum dot (QD) coupled to DNA molecules. Coupling with DNAs, the linear optical responses of the peptide QDs will be enhanced significantly in the simultaneous presence of two optical fields. Based on this technique, we propose a scheme to measure the vibrational frequency of DNA and the coupling strength between peptide QD and DNA in all-optical domain. Distinct with metallic quantum dot, biological QD is non-toxic and pollution-free to environment, which will contribute to clinical medicine experiments. This article leads people to know more about the optical behaviors of DNAs-quantum dot system, with the currently popular pump-probe technique. PMID:22340277

  13. Coherent optical spectroscopy in a biological semiconductor quantum dot-DNA hybrid system.

    Science.gov (United States)

    Li, Jin-Jin; Zhu, Ka-Di

    2012-02-16

    We theoretically investigate coherent optical spectroscopy of a biological semiconductor quantum dot (QD) coupled to DNA molecules. Coupling with DNAs, the linear optical responses of the peptide QDs will be enhanced significantly in the simultaneous presence of two optical fields. Based on this technique, we propose a scheme to measure the vibrational frequency of DNA and the coupling strength between peptide QD and DNA in all-optical domain. Distinct with metallic quantum dot, biological QD is non-toxic and pollution-free to environment, which will contribute to clinical medicine experiments. This article leads people to know more about the optical behaviors of DNAs-quantum dot system, with the currently popular pump-probe technique.

  14. Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH.

    Science.gov (United States)

    Komosa, Martin; Root, Heather; Meyn, M Stephen

    2015-02-27

    Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain 300), range widely in length (200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.

  15. Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates.

    Science.gov (United States)

    Lonkar, Pallavi; Dedon, Peter C

    2011-05-01

    Chronic inflammation has long been recognized as a risk factor for many human cancers. One mechanistic link between inflammation and cancer involves the generation of nitric oxide, superoxide and other reactive oxygen and nitrogen species by macrophages and neutrophils that infiltrate sites of inflammation. Although pathologically high levels of these reactive species cause damage to biological molecules, including DNA, nitric oxide at lower levels plays important physiological roles in cell signaling and apoptosis. This raises the question of inflammation-induced imbalances in physiological and pathological pathways mediated by chemical mediators of inflammation. At pathological levels, the damage sustained by nucleic acids represents the full spectrum of chemistries and likely plays an important role in carcinogenesis. This suggests that DNA damage products could serve as biomarkers of inflammation and oxidative stress in clinically accessible compartments such as blood and urine. However, recent studies of the biotransformation of DNA damage products before excretion point to a weakness in our understanding of the biological fates of the DNA lesions and thus to a limitation in the use of DNA lesions as biomarkers. This review will address these and other issues surrounding inflammation-mediated DNA damage on the road to cancer.

  16. Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.

    Science.gov (United States)

    Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei

    2014-07-01

    A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect.

  17. Quantitative changes in sets of proteins as markers of biological response

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, C.S.; Taylor, J.; Gemmell, M.A.; Tollaksen, S.L. (Argonne National Lab., IL (USA)); Lalwani, N.D.; Reddy, J.K. (Northwestern Univ., Chicago, IL (USA))

    1990-01-01

    Exposure to either physical or chemical insults triggers a cascade of bio-chemical events within the target cell. This response requires adjustment within the protein population of the cell, some proteins becoming more abundant (those involved in the cellular response), others less abundant (those not required or counterproductive to the response). Thus, quantitative changes in the global protein population of an exposed biological system may well serve as an indicator of exposure, provided the alterations observed are selective and dose-dependent. In this paper we present results from a study in which liver protein changes induced by exposure of mice to chemicals known to cause peroxisome proliferation and subsequent hepatocellular carcinoma where monitored. Clofibrate, and its chemical analog ciprofibrate, are hypolipidemic drugs. Di-(ethylhexyl)phthalate (DEHP) is a plasticizer used widely in disposable containers for blood products. WY-14643 is a chemical shown to cause hypolipidemic and peroxisome proliferation, similar to clofibrate, ciprofibrate and DEHP, but structurally different from these three chemicals. Thus, two of the four chemicals are structurally similar while the remaining two are very distinct, although all four chemicals cause the same gross biological response. Our results show that although common protein effects are observed in mice exposed to these chemicals, each chemical also causes specific alterations in selective subsets of proteins that could serve as markers of a particular exposure. 13 refs., 4 figs., 1 tab.

  18. A comparison of quantitative reconstruction techniques for PIXE-tomography analysis applied to biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Beasley, D.G., E-mail: dgbeasley@ctn.ist.utl.pt [IST/C2TN, Universidade de Lisboa, Campus Tecnológico e Nuclear, E.N.10, 2686-953 Sacavém (Portugal); Alves, L.C. [IST/C2TN, Universidade de Lisboa, Campus Tecnológico e Nuclear, E.N.10, 2686-953 Sacavém (Portugal); Barberet, Ph.; Bourret, S.; Devès, G.; Gordillo, N.; Michelet, C. [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Le Trequesser, Q. [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Institut de Chimie de la Matière Condensée de Bordeaux (ICMCB, UPR9048) CNRS, Université de Bordeaux, 87 avenue du Dr. A. Schweitzer, Pessac F-33608 (France); Marques, A.C. [IST/IPFN, Universidade de Lisboa, Campus Tecnológico e Nuclear, E.N.10, 2686-953 Sacavém (Portugal); Seznec, H. [Univ. Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Silva, R.C. da [IST/IPFN, Universidade de Lisboa, Campus Tecnológico e Nuclear, E.N.10, 2686-953 Sacavém (Portugal)

    2014-07-15

    The tomographic reconstruction of biological specimens requires robust algorithms, able to deal with low density contrast and low element concentrations. At the IST/ITN microprobe facility new GPU-accelerated reconstruction software, JPIXET, has been developed, which can significantly increase the speed of quantitative reconstruction of Proton Induced X-ray Emission Tomography (PIXE-T) data. It has a user-friendly graphical user interface for pre-processing, data analysis and reconstruction of PIXE-T and Scanning Transmission Ion Microscopy Tomography (STIM-T). The reconstruction of PIXE-T data is performed using either an algorithm based on a GPU-accelerated version of the Maximum Likelihood Expectation Maximisation (MLEM) method or a GPU-accelerated version of the Discrete Image Space Reconstruction Algorithm (DISRA) (Sakellariou (2001) [2]). The original DISRA, its accelerated version, and the MLEM algorithm, were compared for the reconstruction of a biological sample of Caenorhabditis elegans – a small worm. This sample was analysed at the microbeam line of the AIFIRA facility of CENBG, Bordeaux. A qualitative PIXE-T reconstruction was obtained using the CENBG software package TomoRebuild (Habchi et al. (2013) [6]). The effects of pre-processing and experimental conditions on the elemental concentrations are discussed.

  19. Mapping Biological Networks from Quantitative Data-Independent Acquisition Mass Spectrometry: Data to Knowledge Pipelines.

    Science.gov (United States)

    Crowgey, Erin L; Matlock, Andrea; Venkatraman, Vidya; Fert-Bober, Justyna; Van Eyk, Jennifer E

    2017-01-01

    Data-independent acquisition mass spectrometry (DIA-MS) strategies and applications provide unique advantages for qualitative and quantitative proteome probing of a biological sample allowing constant sensitivity and reproducibility across large sample sets. These advantages in LC-MS/MS are being realized in fundamental research laboratories and for clinical research applications. However, the ability to translate high-throughput raw LC-MS/MS proteomic data into biological knowledge is a complex and difficult task requiring the use of many algorithms and tools for which there is no widely accepted standard and best practices are slowly being implemented. Today a single tool or approach inherently fails to capture the full interpretation that proteomics uniquely supplies, including the dynamics of quickly reversible chemically modified states of proteins, irreversible amino acid modifications, signaling truncation events, and, finally, determining the presence of protein from allele-specific transcripts. This chapter highlights key steps and publicly available algorithms required to translate DIA-MS data into knowledge.

  20. Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

    DEFF Research Database (Denmark)

    Fazio, Alessandro

    set of growth dependent genes by using a multi-factorial experimental design. Moreover, new insights into the metabolic response and transcriptional regulation of these genes have been provided by using systems biology tools (Chapter 3). One of the prerequisite of systems biology should......The yeast Saccharomyces cerevisiae is a model organism in biology, being widely used in fundamental research, the first eukaryotic organism to be fully sequenced and the platform for the development of many genomics techniques. Therefore, it is not surprising that S. cerevisiae has also been widely...... used in the field of systems biology during the last decade. This thesis investigates S. cerevisiae growth physiology and DNA damage response by using a systems biology approach. Elucidation of the relationship between growth rate and gene expression is important to understand the mechanisms regulating...

  1. Biological Evidence Management for DNA Analysis in Cases of Sexual Assault

    Directory of Open Access Journals (Sweden)

    Teresa Magalhães

    2015-01-01

    Full Text Available Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody. Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis.

  2. Biological Evidence Management for DNA Analysis in Cases of Sexual Assault.

    Science.gov (United States)

    Magalhães, Teresa; Dinis-Oliveira, Ricardo Jorge; Silva, Benedita; Corte-Real, Francisco; Nuno Vieira, Duarte

    2015-01-01

    Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody). Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis.

  3. Biological Evidence Management for DNA Analysis in Cases of Sexual Assault

    Science.gov (United States)

    Magalhães, Teresa; Dinis-Oliveira, Ricardo Jorge; Silva, Benedita; Corte-Real, Francisco; Nuno Vieira, Duarte

    2015-01-01

    Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody). Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis. PMID:26587562

  4. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidende in rodent bioassays

    NARCIS (Netherlands)

    Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; Bladeren, van P.J.; Rietjens, I.

    2011-01-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate w

  5. Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidende in rodent bioassays

    NARCIS (Netherlands)

    Paini, A.; Scholz, G.; Marin-Kuan, M.; Schilter, B.; O'Brien, J.; Bladeren, van P.J.; Rietjens, I.

    2011-01-01

    This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate

  6. Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

    NARCIS (Netherlands)

    Vaissiere, T.; Cuenin, C.; Paliwal, A.; Vineis, P.; Hoek, G.; Krzyzanowski, M.; Airoldi, L.; Dunning, A.; Garte, S.; Malaveille, C.; Overvad, K.; Clavel-Chapelon, F.; Linseisen, J.; Boeing, H.; Trichopoulou, A.; Trichopoulous, D.; Kaladidi, A.; Palli, D.; Krogh, V.; Tumino, R.; Panico, S.; Bueno de Mesquita, H.B.; Peeters, P.H.M.; Kumle, M.; Gonzalez, C.A.; Martinez, C.; Dorronsoro, M.; Barricarte, A.; Navarro, C.; Quiros, J.R.; Berglund, B.; Janzon, L.; Jarvholm, B.; Day, N.E.; Key, T.J.; Saracci, R.; Kaaks, R.; Riboli, E.; Hainaut, P.; Herceg, Z.

    2009-01-01

    Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has prov

  7. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  8. Biologic

    CERN Document Server

    Kauffman, L H

    2002-01-01

    In this paper we explore the boundary between biology and the study of formal systems (logic). In the end, we arrive at a summary formalism, a chapter in "boundary mathematics" where there are not only containers but also extainers ><, entities open to interaction and distinguishing the space that they are not. The boundary algebra of containers and extainers is to biologic what boolean algebra is to classical logic. We show how this formalism encompasses significant parts of the logic of DNA replication, the Dirac formalism for quantum mechanics, formalisms for protein folding and the basic structure of the Temperley Lieb algebra at the foundations of topological invariants of knots and links.

  9. Cruciform structures are a common DNA feature important for regulating biological processes

    Directory of Open Access Journals (Sweden)

    Arrowsmith Cheryl

    2011-08-01

    Full Text Available Abstract DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werner's syndrome and others. Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIβ, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a the junction-resolving enzymes, (b DNA repair proteins and transcription factors, (c proteins involved in replication and (d chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.

  10. Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

    Science.gov (United States)

    Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

    2016-03-01

    The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

  11. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    Science.gov (United States)

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

  12. Quantitation of cis-diamminedichloroplatinum II (cisplatin)-DNA-intrastrand adducts in testicular and ovarian cancer patients receiving cisplatin chemotherapy.

    Science.gov (United States)

    Reed, E; Yuspa, S H; Zwelling, L A; Ozols, R F; Poirier, M C

    1986-02-01

    The antitumor activity of cis-diamminedichloroplatinum II (cisplatin) is believed to be related to its covalent interaction with DNA where a major DNA binding product is an intrastrand N7-bidentate adduct on adjacent deoxyguanosines. A novel immunoassay was used to quantitate this adduct in buffy coat DNA from testicular and ovarian cancer patients undergoing cisplatin therapy. 44 out of 120 samples taken from 45 cisplatin patients had detectable cisplatin-DNA adducts. No adducts were detected in 18 samples of DNA taken from normal controls, patients on other chemotherapy, or patients before treatment. The quantity of measurable adducts increased as a function of cumulative dose of cisplatin. This was observed both during repeated daily infusion of the drug and over long-term, repeated 21-28 d cycles of administration. These results suggested that adduct removal is slow even though the tissue has a relatively rapid turnover. Patients receiving cisplatin for the first time on 56-d cycles, and those given high doses of cisplatin as a "salvage" regimen, did not accumulate adducts as rapidly as patients on first time chemotherapy on 21- or 28-d cycles. Disease response data, evaluated for 33 cisplatin-treated patients, showed a positive correlation between the formation of DNA adducts and response to drug therapy. However, more data will be required to confirm this relationship. These data show that specific immunological probes can readily be applied to quantitate DNA adducts in patients undergoing cancer chemotherapy.

  13. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    Science.gov (United States)

    Yu, L. D.; Wongkham, W.; Prakrajang, K.; Sangwijit, K.; Inthanon, K.; Thongkumkoon, P.; Wanichapichart, P.; Anuntalabhochai, S.

    2013-06-01

    Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

  14. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wongkham, W. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K.; Inthanon, K. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wanichapichart, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Membrane Science and Technology Research Center, Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkla 90112 (Thailand); Anuntalabhochai, S. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2013-06-15

    Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

  15. SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple Biological Samples.

    Science.gov (United States)

    Cuomo, Alessandro; Soldi, Monica; Bonaldi, Tiziana

    2017-01-01

    Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented "shotgun" workflow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.

  16. Intermolecular Coulombic Decay in Biology: The Initial Electron Detachment from FADH(-) in DNA Photolyases.

    Science.gov (United States)

    Harbach, Philipp H P; Schneider, Matthias; Faraji, Shirin; Dreuw, Andreas

    2013-03-21

    Intermolecular coulombic decay (ICD) is an efficient mechanism of low-energy electron generation in condensed phases and is discussed as their potential source in living cells, tissues, and materials. The first example of ICD as an operating mechanism in real biological systems, that is, in the DNA repair enzymes photolyases, is presented. Photolyase function involves light-induced electron detachment from a reduced flavin adenine dinucleotide (FADH(-)), followed by its transfer to the DNA-lesion triggering repair of covalently bound nucleobase dimers. Modern quantum chemical methods are employed to demonstrate that the transferred electron is efficiently generated via a resonant ICD process between the antenna pigment and the FADH(-) cofactors.

  17. Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

    Institute of Scientific and Technical Information of China (English)

    Ming Shi; Yong Zhang; Ying-Hua Zhu; Jing Zhang; Wei-Jia Xu

    2008-01-01

    AIM: To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

  18. Quantitative generalized ratiometric fluorescence spectroscopy for turbid media based on probe encapsulated by biologically localized embedding

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Xiu-Fang; Chen, Zeng-Ping, E-mail: zpchen2002@hotmail.com; Cui, Yin-Yin; Hu, Yuan-Liang; Yu, Ru-Qin

    2016-05-19

    PEBBLE (probe encapsulated by biologically localized embedding) nanosensor encapsulating an intensity-based fluorescence indicator and an inert reference fluorescence dye inside the pores of stable matrix can be used as a generalized wavelength-ratiometric probe. However, the lack of an efficient quantitative model render the choices of inert reference dyes and intensity-based fluorescence indicators used in PEBBLEs based generalized wavelength-ratiometric probes rather limited. In this contribution, an extended quantitative fluorescence model was derived specifically for generalized wavelength-ratiometric probes based on PEBBLE technique (QFM{sub GRP}) with a view to simplify the design of PEBBLEs and hence further extend their application potentials. The effectiveness of QFM{sub GRP} has been tested on the quantitative determination of free Ca{sup 2+} in both simulated and real turbid media using a Ca{sup 2+} sensitive PEBBLE nanosensor encapsulating Rhod-2 and eosin B inside the micropores of stable polyacrylamide matrix. Experimental results demonstrated that QFM{sub GRP} could realize precise and accurate quantification of free Ca{sup 2+} in turbid samples, even though there is serious overlapping between the fluorescence excitation peaks of eosin B and Ca{sup 2+} bound Rhod-2. The average relative predictive error value of QFM{sub GRP} for the test simulated turbid samples was 5.9%, about 2–4 times lower than the corresponding values of partial least squares calibration model and the empirical ratiometric model based on the ratio of fluorescence intensities at the excitation peaks of Ca{sup 2+} bound Rhod-2 and eosin B. The recovery rates of QFM{sub GRP} for the real and spiked turbid samples varied from 93.1% to 101%, comparable to the corresponding results of atomic absorption spectrometry. - Highlights: • An advanced model was derived for generalized wavelength-ratiometric PEBBLEs. • The model can simplify the design of generalized wavelength

  19. Artefatos biológicos no EEG quantitativo Biologic artifacts in quantitative EEG

    Directory of Open Access Journals (Sweden)

    Renato Anghinah

    2006-06-01

    Full Text Available Estudamos, em 10 indivíduos adultos normais, o comportamento de cinco artefatos biológicos do eletrencefalograma (EEG: piscamento palpebral, fechamento forçado dos olhos, fechamento forçado da mandíbula, movimentos de língua e varredura horizontal dos olhos - tanto por análise visual como espectral - tanto com objetivo de verificar como esses artefatos são visualizados quando apresentados em mapas de potência da amplitude espectral. Observamos que os potenciais do espectro respeitavam a mesma disposição topográfica que os encontrados à análise visual do traçado. A análise visual do EEG é superior à quantitativa, para o reconhecimento de artefatos, porque preserva a visualização morfológica dos grafoelementos que deve ser feita obrigatoriamente no domínio do tempo, pois a sua correta identificação se perde no domínio da frequência. Devido a grande dificuldade de excluirmos totalmente os artefatos durante o registro do EEG e, por conseguinte, serem incluídos na análise quantitativa, é fundamental conhecermos como estes potenciais serão representados nos mapas quantitativos, para podermos identifica-los, evitando confundí-los com atividades patológicas do EEG.We studied the influence of five biologic artifacts sources on quantitative EEG (blinking, forced eyes closure, forced jaw closure, tongue movements and pursuit eyes movements through both visual and spectral analysis, with the purpose of verifying how do these artifacts can be seen in a cartographic way. We found that the spectrum’s potencials showed the same topographic display that was found through visual analysis. Visual analysis was superior than the quantitative evaluation to recognise the artifacts, as the former preserved the morphological display of the paroxisms. However it is important know how do the potencials are represented in quantitative maps, so that they can be identified as artifacts and not as pathologic EEG activity.

  20. Geant4-DNA simulation of DNA damage caused by direct and indirect radiation effects and comparison with biological data.

    Science.gov (United States)

    Villagrasa, Carmen; Meylan, Sylvain; Gonon, Geraldine; Gruel, Gaëtan; Giesen, Ulrich; Bueno, Marta; Rabus, Hans

    2017-09-01

    In this work we present results obtained in the frame of the BioQuaRT project. The objective of the study was the correlation between the number of radiation-induced double strand breaks (DSB) of the DNA molecule and the probability of detecting nuclear foci after targeted microbeam irradiation of cells with protons and alpha particles of different LET. The former were obtained by simulation with new methods integrated into Geant4-DNA that permit calculating the number of DSB in a DNA target model induced by direct and indirect radiation effects. A particular focus was laid in this work on evaluating the influence of different criteria applied to the simulated results for predicting the formation of a direct SSB. Indeed, these criteria have an important impact on the predicted number of DSB per particle track and its dependence with LET. Among the criteria tested in this work, the case that a direct radiation interaction leads to a strand break if the cumulative energy deposited in the backbone part of one nucleotide exceeds a threshold of 17.5 eV leads to the best agreement with the relative LET dependence of number of radiation induced foci. Further calculations and experimental data are nevertheless needed in order to fix the simulation parameters and to help interpreting the biological experimental data observed by immunofluorescence in terms of the DSB complexity.

  1. Monitoring intracellular polyphosphate accumulation in enhanced biological phosphorus removal systems by quantitative image analysis.

    Science.gov (United States)

    Mesquita, Daniela P; Amaral, A Luís; Leal, Cristiano; Carvalheira, Mónica; Cunha, Jorge R; Oehmen, Adrian; Reis, Maria A M; Ferreira, Eugénio C

    2014-01-01

    A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.

  2. Antiproliferative Pt(IV) complexes: synthesis, biological activity, and quantitative structure-activity relationship modeling.

    Science.gov (United States)

    Gramatica, Paola; Papa, Ester; Luini, Mara; Monti, Elena; Gariboldi, Marzia B; Ravera, Mauro; Gabano, Elisabetta; Gaviglio, Luca; Osella, Domenico

    2010-09-01

    Several Pt(IV) complexes of the general formula [Pt(L)2(L')2(L'')2] [axial ligands L are Cl-, RCOO-, or OH-; equatorial ligands L' are two am(m)ine or one diamine; and equatorial ligands L'' are Cl- or glycolato] were rationally designed and synthesized in the attempt to develop a predictive quantitative structure-activity relationship (QSAR) model. Numerous theoretical molecular descriptors were used alongside physicochemical data (i.e., reduction peak potential, Ep, and partition coefficient, log Po/w) to obtain a validated QSAR between in vitro cytotoxicity (half maximal inhibitory concentrations, IC50, on A2780 ovarian and HCT116 colon carcinoma cell lines) and some features of Pt(IV) complexes. In the resulting best models, a lipophilic descriptor (log Po/w or the number of secondary sp3 carbon atoms) plus an electronic descriptor (Ep, the number of oxygen atoms, or the topological polar surface area expressed as the N,O polar contribution) is necessary for modeling, supporting the general finding that the biological behavior of Pt(IV) complexes can be rationalized on the basis of their cellular uptake, the Pt(IV)-->Pt(II) reduction, and the structure of the corresponding Pt(II) metabolites. Novel compounds were synthesized on the basis of their predicted cytotoxicity in the preliminary QSAR model, and were experimentally tested. A final QSAR model, based solely on theoretical molecular descriptors to ensure its general applicability, is proposed.

  3. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.

    Science.gov (United States)

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-08-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil(®), PowerFecal(®), NucleoSpin(®) Soil kits and QIAamp(®) DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin(®) Soil kit (NS kit), and to a lesser extent the PowerFecal(®) kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. Real-time polymerase chain reaction approach for quantitation of ruminant-specific DNA to indicate a correlation between DNA amount and meat and bone meal heat treatments.

    Science.gov (United States)

    Chiappini, Barbara; Brambilla, Gianfranco; Agrimi, Umberto; Vaccari, Gabriele; Aarts, Henk J M; Berben, Gilbert; Frezza, Domenico; Giambra, Vincenzo

    2005-01-01

    The use of ruminant-derived proteins in ruminant feeds has been banned in both the European Union and the United States to prevent further spread of bovine spongiform encephalopathy. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in feed. We developed a quantitative real-time polymerase chain reaction assay for the quantification of ruminant-specific DNA as index of protein content. The assay is based on the amplification of a 117 base pair mitochondrial 16S rRNA DNA gene fragment and an internal positive control (IPC). The use of an IPC permits compensation for differences in DNA extraction efficiency and avoids the occurrence of false-negative results. We demonstrated a decrease in target DNA amount with a difference of 2 logs between meat and bone meal (MBM) treated at 133 degrees and 145 degrees C. Such a difference indicates that bias could occur when DNA-based methods are used for quantitation purposes. Risk management could benefit from future efforts concerning validation of the method for MBM detection in feedstuff and safety evaluation of the use of animal-derived proteins in animal nutrition.

  5. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA.

    Science.gov (United States)

    Hunter, Margaret E; Dorazio, Robert M; Butterfield, John S S; Meigs-Friend, Gaia; Nico, Leo G; Ferrante, Jason A

    2017-03-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low-concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species' presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty-indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  6. 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.

  7. Biological effects induced by K photo-ionisation in and near constituent atoms of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Touati, A.; Herve du Penhoot, M.A.; Fayard, B.; Champion, C.; Abel, F.; Gobert, F.; Lamoureux, M.; Politis, M.F.; Martins, L.; Ricoul, M.; Sabatier, L.; Sage, E.; Chetioui, A

    2002-07-01

    In order to assess the lethal efficiency and other biological effects of inner shell ionisations of constituent atoms of DNA ('K' events), experiments were developed at the LURE synchrotron facility using ultrasoft X rays as a probe of K events. The lethal efficiency of ultrasoft X rays above the carbon K threshold was especially investigated using V79 cells and compared with their efficiency to induce double strand breaks in dry plasmid-DNA. A correlation between the K event efficiencies for these processes is shown. Beams of 340 eV were found to be twice as efficient at killing cells than were beams at 250 eV. In addition, a rough two-fold increase of the relative biological effectiveness for dicentric+ring induction has also been observed between 250 and 340 eV radiations. (author)

  8. The B- to Z-DNA equilibrium in vivo is perturbed by biological processes.

    OpenAIRE

    Zacharias, W; Jaworski, A; Larson, J E; Wells, R D

    1988-01-01

    Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli. The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes. The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo. The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for imp...

  9. How selection fashions morphological variation in Cakile maritima: A comparative analysis of population structure using random amplified polymorphic DNA and quantitative traits

    Institute of Scientific and Technical Information of China (English)

    Gandour MHEMMED; Hessini KAMEL; Abdelly CHEDLY

    2012-01-01

    It is a long-standing debate in evolutionary biology whether natural selection can generate divergence in the face of gene flow.Comparative studies of quantitative genetic and neutral marker differentiation have provided means for detecting the action of selection and random genetic drift in natural populations.We estimated the degree of population divergence in several quantitative traits and compared these estimates with that based on presumably neutral molecular markers (random amplified polymorphic DNA [RAPD]).This approach allowed us to disentangle the effects of divergent selection from that of other evolutionary forces.Nine populations of Cakile maritima,which encompasses the complete range of distribution of this species in Tunisia,were examined.We found a high proportion of total genetic variance to be among populations and among ecoregions for quantitative traits (range of QsT:0.44-0.88) and a moderate one for RAPD markers (GsT:0.081).In addition,almost all characters displayed a significant higher QsT than GsT,indicating occurrence of phenotypic plasticity and local adaptation.The latter is explicable as there is no reason to expect that natural selection would affect in similar fashion all traits and affect all populations at a similar level.We also found a negative and significant correlation between genetic variation in molecular marker loci and quantitative traits at the multitrait scale.This result attests that the evolution of these markers in C.maritima were not paralleled,suggesting that the degree of genetic differentiation in neutral marker loci is closely predictive of the degree of differentiation in loci coding quantitative traits and the majority of these neutral markers negatively controlled the studied quantitative traits.

  10. Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus

    Science.gov (United States)

    Gorički, Špela; Stanković, David; Snoj, Aleš; Kuntner, Matjaž; Jeffery, William R.; Trontelj, Peter; Pavićević, Miloš; Grizelj, Zlatko; Năpăruş-Aljančič, Magdalena; Aljančič, Gregor

    2017-03-01

    Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

  11. Quantitative Modeling of Membrane Transport and Anisogamy by Small Groups Within a Large-Enrollment Organismal Biology Course

    Directory of Open Access Journals (Sweden)

    Eric S. Haag

    2016-12-01

    Full Text Available Quantitative modeling is not a standard part of undergraduate biology education, yet is routine in the physical sciences. Because of the obvious biophysical aspects, classes in anatomy and physiology offer an opportunity to introduce modeling approaches to the introductory curriculum. Here, we describe two in-class exercises for small groups working within a large-enrollment introductory course in organismal biology. Both build and derive biological insights from quantitative models, implemented using spreadsheets. One exercise models the evolution of anisogamy (i.e., small sperm and large eggs from an initial state of isogamy. Groups of four students work on Excel spreadsheets (from one to four laptops per group. The other exercise uses an online simulator to generate data related to membrane transport of a solute, and a cloud-based spreadsheet to analyze them. We provide tips for implementing these exercises gleaned from two years of experience.

  12. Quantitative global and gene-specific promoter methylation in relation to biological properties of neuroblastomas

    Directory of Open Access Journals (Sweden)

    Kiss Nimrod B

    2012-09-01

    Full Text Available Abstract Background In this study we aimed to quantify tumor suppressor gene (TSG promoter methylation densities levels in primary neuroblastoma tumors and cell lines. A subset of these TSGs is associated with a CpG island methylator phenotype (CIMP in other tumor types. Methods The study panel consisted of 38 primary tumors, 7 established cell lines and 4 healthy references. Promoter methylation was determined by bisulphate Pyrosequencing for 14 TSGs; and LINE-1 repeat element methylation was used as an indicator of global methylation levels. Results Overall mean TSG Z-scores were significantly increased in cases with adverse outcome, but were unrelated to global LINE-1 methylation. CIMP with hypermethylation of three or more gene promoters was observed in 6/38 tumors and 7/7 cell lines. Hypermethylation of one or more TSG (comprising TSGs BLU, CASP8, DCR2, CDH1, RASSF1A and RASSF2 was evident in 30/38 tumors. By contrast only very low levels of promoter methylation were recorded for APC, DAPK1, NORE1A, P14, P16, TP73, PTEN and RARB. Similar involvements of methylation instability were revealed between cell line models and neuroblastoma tumors. Separate analysis of two proposed CASP8 regulatory regions revealed frequent and significant involvement of CpG sites between exon 4 and 5, but modest involvement of the exon 1 region. Conclusions/significance The results highlight the involvement of TSG methylation instability in neuroblastoma tumors and cell lines using quantitative methods, support the use of DNA methylation analyses as a prognostic tool for this tumor type, and underscore the relevance of developing demethylating therapies for its treatment.

  13. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

    Directory of Open Access Journals (Sweden)

    Dominika Żurek-Biesiada

    2016-06-01

    Full Text Available Single Molecule Localization Microscopy (SMLM is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015 [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.

  14. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.

    Science.gov (United States)

    Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

    2016-06-01

    Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.

  15. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    2012-01-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal...... samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six...... different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples...

  16. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study DNA was extracted either directly or following freeze storage of three homogenized human fecal...... samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples, however differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different...... primer pairs targeting 16S rRNA genes of significant bacterial groups and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven out of nine cases the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal-samples that had...

  17. The Science and Issues of Human DNA Polymorphisms: A Training Workshop for High School Biology Teachers

    Energy Technology Data Exchange (ETDEWEB)

    Micklos, David A.

    2006-10-30

    This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms â which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, and computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nationâÂÂs oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human

  18. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  19. A semi-quantitative assay of overall DNA methylation status using Methyl-CpG binding protein (MBD1

    Directory of Open Access Journals (Sweden)

    Zhang Chunxiao

    2012-05-01

    Full Text Available Abstract Background In mammals, DNA methylation at the 5-position of cytosine is the most essential epigenetic modification. Changes in the level of genome-wide DNA methylation (also known as overall DNA methylation are associated with alterations in gene expression, thereby contributing to the phenotypic and physiological diversity. Current technologies for detecting overall DNA methylation either suffer from low sensitivity or require sophisticated equipment. Studies on domestic animals are hampered by the lack of complete and annotated genomic information. Results Here we report a rapid slot blot method using methyl-CpG binding protein (MBD1 to exam the level of overall DNA methylation in pigs and chickens. Using this rapid approach, we determined the methylation status in various DNA samples of a Chinese indigenous (Erhualian and a Western (Large White breed of pigs. We also chose day 18 embryos (E18 and newly hatched chicks (D1 of a Chinese indigenous chicken breed (Wen’s yellow-feathered broiler chicken for genome-wide DNA methylation analysis. The results revealed tissue- and breed-specific differences, as well as age-dependent variations, in the level of overall DNA methylation. Conclusion The results showed that the slot blot assay is a sensitive, highly specific and convenient method for semi-quantitative estimation of overall DNA methylation with no species specificity. This method does not require sophisticated equipment, such as high performance liquid chromatography (HPLC, or expensive technologies like sequencing, thus providing a useful tool for overall DNA methylation studies on domestic animals.

  20. Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.

    Science.gov (United States)

    Peper, Grit; Fankhauser, Alexander; Merlin, Thomas; Roscic, Ana; Hofmann, Matthias; Obrdlik, Petr

    2014-11-01

    Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.

  1. Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR.

    Science.gov (United States)

    Fykse, Else Marie; Olsen, Jaran Strand; Skogan, Gunnar

    2003-10-01

    A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

  2. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  3. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  4. Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method

    Directory of Open Access Journals (Sweden)

    Sette Alessandro

    2005-05-01

    Full Text Available Abstract Background Many processes in molecular biology involve the recognition of short sequences of nucleic-or amino acids, such as the binding of immunogenic peptides to major histocompatibility complex (MHC molecules. From experimental data, a model of the sequence specificity of these processes can be constructed, such as a sequence motif, a scoring matrix or an artificial neural network. The purpose of these models is two-fold. First, they can provide a summary of experimental results, allowing for a deeper understanding of the mechanisms involved in sequence recognition. Second, such models can be used to predict the experimental outcome for yet untested sequences. In the past we reported the development of a method to generate such models called the Stabilized Matrix Method (SMM. This method has been successfully applied to predicting peptide binding to MHC molecules, peptide transport by the transporter associated with antigen presentation (TAP and proteasomal cleavage of protein sequences. Results Herein we report the implementation of the SMM algorithm as a publicly available software package. Specific features determining the type of problems the method is most appropriate for are discussed. Advantageous features of the package are: (1 the output generated is easy to interpret, (2 input and output are both quantitative, (3 specific computational strategies to handle experimental noise are built in, (4 the algorithm is designed to effectively handle bounded experimental data, (5 experimental data from randomized peptide libraries and conventional peptides can easily be combined, and (6 it is possible to incorporate pair interactions between positions of a sequence. Conclusion Making the SMM method publicly available enables bioinformaticians and experimental biologists to easily access it, to compare its performance to other prediction methods, and to extend it to other applications.

  5. Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens.

    Science.gov (United States)

    Aronova, M A; Kim, Y C; Pivovarova, N B; Andrews, S B; Leapman, R D

    2009-02-01

    Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path lambda. By acquiring pairs of images, one above and one below the Ca L(2,3) edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/lambda) map, it is possible to correct the Ca L(2,3) signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 microm2 areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2 x 10(-4). Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.

  6. Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens

    Energy Technology Data Exchange (ETDEWEB)

    Aronova, M.A. [Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bldg. 13, Rm. 3N17, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Kim, Y.C. [Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Pivovarova, N.B.; Andrews, S.B. [Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (United States); Leapman, R.D. [Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bldg. 13, Rm. 3N17, 9000 Rockville Pike, Bethesda, MD 20892 (United States)], E-mail: leapmanr@mail.nih.gov

    2009-02-15

    Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path {lambda}. By acquiring pairs of images, one above and one below the Ca L{sub 2,3} edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/{lambda}) map, it is possible to correct the Ca L{sub 2,3} signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 {mu}m{sup 2} areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2x10{sup -4}. Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.

  7. Mammographic quantitative image analysis and biologic image composition for breast lesion characterization and classification

    Energy Technology Data Exchange (ETDEWEB)

    Drukker, Karen, E-mail: kdrukker@uchicago.edu; Giger, Maryellen L.; Li, Hui [Department of Radiology, University of Chicago, Chicago, Illinois 60637 (United States); Duewer, Fred; Malkov, Serghei; Joe, Bonnie; Kerlikowske, Karla; Shepherd, John A. [Radiology Department, University of California, San Francisco, California 94143 (United States); Flowers, Chris I. [Department of Radiology, University of South Florida, Tampa, Florida 33612 (United States); Drukteinis, Jennifer S. [Department of Radiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 (United States)

    2014-03-15

    Purpose: To investigate whether biologic image composition of mammographic lesions can improve upon existing mammographic quantitative image analysis (QIA) in estimating the probability of malignancy. Methods: The study population consisted of 45 breast lesions imaged with dual-energy mammography prior to breast biopsy with final diagnosis resulting in 10 invasive ductal carcinomas, 5 ductal carcinomain situ, 11 fibroadenomas, and 19 other benign diagnoses. Analysis was threefold: (1) The raw low-energy mammographic images were analyzed with an established in-house QIA method, “QIA alone,” (2) the three-compartment breast (3CB) composition measure—derived from the dual-energy mammography—of water, lipid, and protein thickness were assessed, “3CB alone”, and (3) information from QIA and 3CB was combined, “QIA + 3CB.” Analysis was initiated from radiologist-indicated lesion centers and was otherwise fully automated. Steps of the QIA and 3CB methods were lesion segmentation, characterization, and subsequent classification for malignancy in leave-one-case-out cross-validation. Performance assessment included box plots, Bland–Altman plots, and Receiver Operating Characteristic (ROC) analysis. Results: The area under the ROC curve (AUC) for distinguishing between benign and malignant lesions (invasive and DCIS) was 0.81 (standard error 0.07) for the “QIA alone” method, 0.72 (0.07) for “3CB alone” method, and 0.86 (0.04) for “QIA+3CB” combined. The difference in AUC was 0.043 between “QIA + 3CB” and “QIA alone” but failed to reach statistical significance (95% confidence interval [–0.17 to + 0.26]). Conclusions: In this pilot study analyzing the new 3CB imaging modality, knowledge of the composition of breast lesions and their periphery appeared additive in combination with existing mammographic QIA methods for the distinction between different benign and malignant lesion types.

  8. A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging

    Science.gov (United States)

    Noller, Crystal M.; Boulina, Maria; McNamara, George; Szeto, Angela; McCabe, Philip M.

    2016-01-01

    Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 μm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines. PMID:27182204

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

    Science.gov (United States)

    Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

    2017-01-01

    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.

  10. 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.

  11. Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR

    Science.gov (United States)

    Holland, Rebecca; Underwood, Sarah; Fairlie, Jennifer; Psifidi, Androniki; Ilska, Joanna J.; Bagnall, Ainsley; Whitelaw, Bruce; Coffey, Mike; Banos, Georgios; Nussey, Daniel H.

    2016-01-01

    Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for

  12. Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

    Science.gov (United States)

    Ajaz, Saima; Czajka, Anna; Malik, Afshan

    2015-01-01

    We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.

  13. Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.

    Science.gov (United States)

    Seeker, Luise A; Holland, Rebecca; Underwood, Sarah; Fairlie, Jennifer; Psifidi, Androniki; Ilska, Joanna J; Bagnall, Ainsley; Whitelaw, Bruce; Coffey, Mike; Banos, Georgios; Nussey, Daniel H

    2016-01-01

    Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for

  14. A quantitative assessment of the Hadoop framework for analyzing massively parallel DNA sequencing data.

    Science.gov (United States)

    Siretskiy, Alexey; Sundqvist, Tore; Voznesenskiy, Mikhail; Spjuth, Ola

    2015-01-01

    New high-throughput technologies, such as massively parallel sequencing, have transformed the life sciences into a data-intensive field. The most common e-infrastructure for analyzing this data consists of batch systems that are based on high-performance computing resources; however, the bioinformatics software that is built on this platform does not scale well in the general case. Recently, the Hadoop platform has emerged as an interesting option to address the challenges of increasingly large datasets with distributed storage, distributed processing, built-in data locality, fault tolerance, and an appealing programming methodology. In this work we introduce metrics and report on a quantitative comparison between Hadoop and a single node of conventional high-performance computing resources for the tasks of short read mapping and variant calling. We calculate efficiency as a function of data size and observe that the Hadoop platform is more efficient for biologically relevant data sizes in terms of computing hours for both split and un-split data files. We also quantify the advantages of the data locality provided by Hadoop for NGS problems, and show that a classical architecture with network-attached storage will not scale when computing resources increase in numbers. Measurements were performed using ten datasets of different sizes, up to 100 gigabases, using the pipeline implemented in Crossbow. To make a fair comparison, we implemented an improved preprocessor for Hadoop with better performance for splittable data files. For improved usability, we implemented a graphical user interface for Crossbow in a private cloud environment using the CloudGene platform. All of the code and data in this study are freely available as open source in public repositories. From our experiments we can conclude that the improved Hadoop pipeline scales better than the same pipeline on high-performance computing resources, we also conclude that Hadoop is an economically viable

  15. Biological chemistry as a foundation of DNA genealogy: the emergence of "molecular history".

    Science.gov (United States)

    Klyosov, A A

    2011-05-01

    This paper presents the basis of DNA genealogy, a new field of science, which is currently emerging as an unusual blend of biochemistry, history, linguistics, and chemical kinetics. The methodology of the new approach is comprised of chemical (biological) kinetics applied to a pattern of mutations in non-recombinant fragments of DNA (Y chromosome and mtDNA, the latter not being considered in this overview). The goal of the analysis is to translate DNA mutation patterns into time spans to the most recent common ancestors of a given population or tribe and to the dating of ancient migration routes. To illustrate this approach, time spans to the common ancestors are calculated for ethnic Russians, that is Eastern Slavs (R1a1 tribe), Western Slavs (I1 and I2 tribes), and Northern (or Uralic) Slavs (N1c tribe), which were found to live around 4600 years before present (R1a1), 3650 ybp (I1), 3000 and 10,500 ybp (I2, two principal DNA lineages), and 3525 ybp (N1c) (confidence intervals are given in the main text). The data were compared with the respective dates for the nearest common ancestor of the R1a1 "Indo-European" population in India, who lived 4050 years before present, whose descendants represent the majority of the upper castes in India today (up to 72%). Furthermore, it was found that the haplotypes of ethnic Russians of the R1a1 haplogroup (up to 62% of the population in the Russian Federation) and those of the R1a1 Indians (more than 100 million today) are practically identical to each other, up to 67-marker haplotypes. This essentially solves a 200-year-old mystery of who were the Aryans who arrived in India around 3500 years before the present. Haplotypes and time spans to the ancient common ancestors were also compared for the ethnic Russians of haplogroups I1 and I2, on one hand, and the respective I1 and I2 populations in Eastern and Western Europe and Scandinavia, on the other. It is suggested that the approach described in this overview lays the

  16. Explicit tracking of uncertainty increases the power of quantitative rule-of-thumb reasoning in cell biology.

    Science.gov (United States)

    Johnston, Iain G; Rickett, Benjamin C; Jones, Nick S

    2014-12-01

    Back-of-the-envelope or rule-of-thumb calculations involving rough estimates of quantities play a central scientific role in developing intuition about the structure and behavior of physical systems, for example in so-called Fermi problems in the physical sciences. Such calculations can be used to powerfully and quantitatively reason about biological systems, particularly at the interface between physics and biology. However, substantial uncertainties are often associated with values in cell biology, and performing calculations without taking this uncertainty into account may limit the extent to which results can be interpreted for a given problem. We present a means to facilitate such calculations where uncertainties are explicitly tracked through the line of reasoning, and introduce a probabilistic calculator called CALADIS, a free web tool, designed to perform this tracking. This approach allows users to perform more statistically robust calculations in cell biology despite having uncertain values, and to identify which quantities need to be measured more precisely to make confident statements, facilitating efficient experimental design. We illustrate the use of our tool for tracking uncertainty in several example biological calculations, showing that the results yield powerful and interpretable statistics on the quantities of interest. We also demonstrate that the outcomes of calculations may differ from point estimates when uncertainty is accurately tracked. An integral link between CALADIS and the BioNumbers repository of biological quantities further facilitates the straightforward location, selection, and use of a wealth of experimental data in cell biological calculations.

  17. Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

    Science.gov (United States)

    Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

    2004-12-01

    Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

  18. Forensic science, genetics and wildlife biology: getting the right mix for a wildlife DNA forensics lab.

    Science.gov (United States)

    Ogden, Rob

    2010-09-01

    Wildlife DNA forensics is receiving increasing coverage in the popular press and has begun to appear in the scientific literature in relation to several different fields. Recognized as an applied subject, it rests on top of very diverse scientific pillars ranging from biochemistry through to evolutionary genetics, all embedded within the context of modern forensic science. This breadth of scope, combined with typically limited resources, has often left wildlife DNA forensics hanging precariously between human DNA forensics and academics keen to seek novel applications for biological research. How best to bridge this gap is a matter for regular debate among the relatively few full-time practitioners in the field. The decisions involved in establishing forensic genetic services to investigate wildlife crime can be complex, particularly where crimes involve a wide range of species and evidential questions. This paper examines some of the issues relevant to setting up a wildlife DNA forensics laboratory based on experiences of working in this area over the past 7 years. It includes a discussion of various models for operating individual laboratories as well as options for organizing forensic testing at higher national and international levels.

  19. DNA binding properties and biological evaluation of dihydropyrimidinones derivatives as potential antitumor agents

    Science.gov (United States)

    Wang, Gongke; Li, Xiangrong; Gou, Yaping; Chen, Yuhan; Yan, Changling; Lu, Yan

    2013-10-01

    The binding properties of two medicinally important dihydropyrimidinones derivatives 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(Ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (EMCD) with calf-thymus DNA (ctDNA) were investigated by spectroscopy, viscosity, isothermal titration calorimetry (ITC) and molecular modeling techniques. Simultaneously, their biological activities were evaluated with MTT assay method. The binding constants determined with spectroscopic titration and ITC were found to be in the same order of 104 M-1. According to the results of viscosity studies, fluorescence competitive binding experiment and ITC investigations, intercalative binding was evaluated as the dominant binding modes between the two compounds and ctDNA. Furthermore, the results of molecular modeling corroborated those obtained from spectroscopic, viscosimetric and ITC investigations. Evaluation of the antitumor activities of the two derivatives against different tumor cell lines proved that they exhibited significant tumor cell inhibition rate, accordingly blocking DNA transcription and replication. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases.

  20. Quantitative correlation of the in vitro biological effect with parameters of molecular complexation in mutagen-interceptor systems.

    Science.gov (United States)

    Buchelnikov, Anatoly S; Evstigneev, Maxim P

    2014-09-21

    According to the theory of interceptor-protector action a quantitative link between the physico-chemical parameters of molecular complexation and in vitro biological effect in aromatic drug-interceptor systems must exist. In the present communication such link between relative change in mutagenicity of IQ-type aromatic mutagens on addition of aromatic interceptor molecules with equilibrium hetero-association constants of mutagen-interceptor complexation has been found using the published in vitro data in bacteria cell systems.

  1. Quantitative redox biology: an approach to understand the role of reactive species in defining the cellular redox environment.

    Science.gov (United States)

    Buettner, Garry R; Wagner, Brett A; Rodgers, Victor G J

    2013-11-01

    Systems biology is now recognized as a needed approach to understand the dynamics of inter- and intra-cellular processes. Redox processes are at the foundation of nearly all aspects of biology. Free radicals, related oxidants, and antioxidants are central to the basic functioning of cells and tissues. They set the cellular redox environment and, therefore, are the key to regulation of biochemical pathways and networks, thereby influencing organism health. To understand how short-lived, quasi-stable species, such as superoxide, hydrogen peroxide, and nitric oxide, connect to the metabolome, proteome, lipidome, and genome we need absolute quantitative information on all redox active compounds as well as thermodynamic and kinetic information on their reactions, i.e., knowledge of the complete redoxome. Central to the state of the redoxome are the interactive details of the superoxide/peroxide formation and removal systems. Quantitative information is essential to establish the dynamic mathematical models needed to reveal the temporal evolution of biochemical pathways and networks. This new field of Quantitative Redox Biology will allow researchers to identify new targets for intervention to advance our efforts to achieve optimal human health.

  2. Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish.

    Science.gov (United States)

    Liu, Jingang; Gong, Lu; Chang, Changqing; Liu, Cong; Peng, Jinrong; Chen, Jun

    2012-09-20

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  3. Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    Jingang Liu; Lu Gong; Changqing Chang; Cong Liu; Jinrong Peng; Jun Chen

    2012-01-01

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce Ⅰ,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination (HR),non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce Ⅰ recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by I-Sce Ⅰ could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseⅣ (lig4) when the NHEJ construct was cut by I-Sce Ⅰ in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  4. Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts 1

    Science.gov (United States)

    Uchimiya, Hirofumi; Murashige, Toshio

    1977-01-01

    After a 5-hour incubation of protoplasts of Nicotiana tabacum L. `Xanthi' with 3H-DNA (7.26 μg/ml) from N. tabacum L. `Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene. A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli3H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the “soluble” fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 × 10−15g donor DNA, or

  5. Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation: Which Is the Best Quantitative Indicator of DNA Degradation?

    Institute of Scientific and Technical Information of China (English)

    LIU Lijiang; SHU Xiji; REN Liang; ZHOU Hongyan; LI Yan; LIU Wei; ZHU Cheng; LIU Liang

    2007-01-01

    This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined the early time of death by computerized image analysis technique (CIAT) and identified the best parameter that quantitatively reflects the DNA degradation.The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within the first 36 h after death, stained by Feulgen-Van's staining, three indices reflecting DNA content in splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray scale (AG) were measured by the image analysis. Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h. A stepwise linear regression analysis showed that only AG was fitted. A correlation between the postmortem interval (PMI) and AG was identified and the corresponding regression equation was obtained. Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility,and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

  6. Bodies of science and law: forensic DNA profiling, biological bodies, and biopower.

    Science.gov (United States)

    Toom, Victor

    2012-01-01

    How is jurisdiction transferred from an individual's biological body to agents of power such as the police, public prosecutors, and the judiciary, and what happens to these biological bodies when transformed from private into public objects? These questions are examined by analysing bodies situated at the intersection of science and law. More specifically, the transformation of ‘private bodies’ into ‘public bodies’ is analysed by going into the details of forensic DNA profiling in the Dutch jurisdiction. It will be argued that various ‘forensic genetic practices’ enact different forensic genetic bodies'. These enacted forensic genetic bodies are connected with various infringements of civil rights, which become articulated in exploring these forensic genetic bodies’‘normative registers’.

  7. New insight into the molecular mechanisms of the biological effects of DNA minor groove binders.

    Directory of Open Access Journals (Sweden)

    Xinbo Zhang

    Full Text Available BACKGROUND: Bisbenzimides, or Hoechst 33258 (H258, and its derivative Hoechst 33342 (H342 are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear. METHODOLOGY/PRINCIPAL FINDINGS: To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects. CONCLUSIONS/SIGNIFICANCE: A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug

  8. The Science and Issues of Human DNA Polymoprhisms: A Training Workshop for High School Biology Teachers

    Energy Technology Data Exchange (ETDEWEB)

    David. A Micklos

    2006-10-30

    This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms – which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, and computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nation’s oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human polymorphism

  9. The Science and Issues of Human DNA Polymorphisms: A Training Workshop for High School Biology Teachers

    Energy Technology Data Exchange (ETDEWEB)

    Micklos, David A.

    2006-10-30

    This project achieved its goal of implementing a nationwide training program to introduce high school biology teachers to the key uses and societal implications of human DNA polymorphisms. The 2.5-day workshop introduced high school biology faculty to a laboratory-based unit on human DNA polymorphisms â which provides a uniquely personal perspective on the science and Ethical, Legal and Social Implications (ELSI) of the Human Genome Project. As proposed, 12 workshops were conducted at venues across the United States. The workshops were attended by 256 high school faculty, exceeding proposed attendance of 240 by 7%. Each workshop mixed theoretical, laboratory, and computer work with practical and ethical implications. Program participants learned simplified lab techniques for amplifying three types of chromosomal polymorphisms: an Alu insertion (PV92), a VNTR (pMCT118/D1S80), and single nucleotide polymorphisms (SNPs) in the mitochondrial control region. These polymorphisms illustrate the use of DNA variations in disease diagnosis, forensic biology, and identity testing - and provide a starting point for discussing the uses and potential abuses of genetic technology. Participants also learned how to use their Alu and mitochondrial data as an entrée to human population genetics and evolution. Our work to simplify lab techniques for amplifying human DNA polymorphisms in educational settings culminated with the release in 1998 of three Advanced Technology (AT) PCR kits by Carolina Biological Supply Company, the nationâÂÂs oldest educational science supplier. The kits use a simple 30-minute method to isolate template DNA from hair sheaths or buccal cells and streamlined PCR chemistry based on Pharmacia Ready-To-Go Beads, which incorporate Taq polymerase, deoxynucleotide triphosphates, and buffer in a freeze-dried pellet. These kits have greatly simplified teacher implementation of human PCR labs, and their use is growing at a rapid pace. Sales of human

  10. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

    2011-05-13

    We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  11. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Science.gov (United States)

    Ali, M. E.; Hashim, U.; Mustafa, S.; Che Man, Y. B.; Yusop, M. H. M.; Bari, M. F.; Islam, Kh N.; Hasan, M. F.

    2011-05-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml - 1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  12. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples.

    Science.gov (United States)

    Ali, M E; Hashim, U; Mustafa, S; Man, Y B Che; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-05-13

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  13. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    Science.gov (United States)

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  14. Quantitative and qualitative assessment of DNA extracted from saliva for its use in forensic identification

    Directory of Open Access Journals (Sweden)

    Parul Khare

    2014-01-01

    Full Text Available Saliva has long been known for its diagnostic value in several diseases. It also has a potential to be used in forensic science. Objective: The objective of this study is to compare the quantity and quality of DNA samples extracted from saliva with those extracted from blood in order to assess the feasibility of extracting sufficient DNA from saliva for its possible use in forensic identification. Materials and Methods: Blood and saliva samples were collected from 20 volunteers and DNA extraction was performed through Phenol Chloroform technique. The quantity and quality of isolated DNA was analyzed by spectrophotometery and the samples were then used to amplify short tandem repeat (STR F13 using the polymerase chain reaction. Results: Mean quantity of DNA obtained in saliva was 48.4 ± 8.2 μg/ml and in blood was 142.5 ± 45.9 μg/ml. Purity of DNA obtained as assessed by the ratio of optical density 260/280, was found to be optimal in 45% salivary samples while remaining showed minor contamination. Despite this positive F13 STR amplification was achieved in 75% of salivary DNA samples. Conclusion: Results of this study showed that saliva may prove to be a useful source of DNA for forensic purpose.

  15. Polycyclic Aromatic Hydrocarbon (PAH Exposure and DNA Adduct Semi-Quantitation in Archived Human Tissues

    Directory of Open Access Journals (Sweden)

    M. Margaret Pratt

    2011-06-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAHs are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products, considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS. These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.

  16. Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

    Directory of Open Access Journals (Sweden)

    Woodfine Kathryn

    2011-01-01

    Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

  17. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-08-24

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

  18. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    Science.gov (United States)

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-12-10

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

  19. CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

    Science.gov (United States)

    Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; Akyürek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Pérez Urquiza, M.; Bautista, J. L.; Garibay, A.; Fuller, B.; Baoutina, A.; Partis, L.; Emslie, K.; Holden, M.; Chum, W. Y.; Kim, H.-H.; Phunbua, N.; Milavec, M.; Zel, J.; Vonsky, M.; Konopelko, L. A.; Lau, T. L. T.; Yang, B.; Hui, M. H. K.; Yu, A. C. H.; Viroonudomphol, D.; Prawettongsopon, C.; Wiangnon, K.; Takabatake, R.; Kitta, K.; Kawaharasaki, M.; Parkes, H.

    2012-01-01

    Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials". The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  20. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  1. DNA assembler: a synthetic biology tool for characterizing and engineering natural product gene clusters.

    Science.gov (United States)

    Shao, Zengyi; Zhao, Huimin

    2012-01-01

    The majority of existing antibacterial and anticancer drugs are natural products or their derivatives. However, the characterization and engineering of these compounds are often hampered by limited ability to manipulate the corresponding biosynthetic pathways. Recently, we developed a genomics-driven, synthetic biology-based method, DNA assembler, for discovery, characterization, and engineering of natural product biosynthetic pathways (Shao, Luo, & Zhao, 2011). By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in individual hosts in a single-step manner. In this chapter, we describe the general guidelines for construct design. By using two distinct biosynthetic pathways, we demonstrate that DNA assembler can perform multiple tasks, including heterologous expression, introduction of single or multiple point mutations, scar-less gene deletion, generation of product derivatives, and creation of artificial gene clusters. As such, this method offers unprecedented flexibility and versatility in pathway manipulations.

  2. Evaluation of the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste from biological research laboratories.

    Science.gov (United States)

    Li, Meng-Nan; Zheng, Guang-Hong; Wang, Lei; Xiao, Wei; Fu, Xiao-Hua; Le, Yi-Quan; Ren, Da-Ming

    2009-01-01

    The discharge of recombinant DNA waste from biological laboratories into the eco-system may be one of the pathways resulting in horizontal gene transfer or "gene pollution". Heating at 100 degrees C for 5-10 min is a common method for treating recombinant DNA waste in biological research laboratories in China. In this study, we evaluated the effectiveness and the safety of the thermo-treatment method in the disposal of recombinant DNA waste. Quantitative PCR, plasmid transformation and electrophoresis technology were used to evaluate the decay/denaturation efficiency during the thermo-treatment process of recombinant plasmid, pET-28b. Results showed that prolonging thermo-treatment time could improve decay efficiency of the plasmid, and its decay half-life was 2.7-4.0 min during the thermo-treatment at 100 degrees C. However, after 30 min of thermo-treatment some transforming activity remained. Higher ionic strength could protect recombinant plasmid from decay during the treatment process. These results indicate that thermo-treatment at 100 degrees C cannot decay and inactivate pET-28b completely. In addition, preliminary results showed that thermo-treated recombinant plasmids were not degraded completely in a short period when they were discharged into an aquatic environment. This implies that when thermo-treated recombinant DNAs are discharged into the eco-system, they may have enough time to re-nature and transform, thus resulting in gene diffusion.

  3. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation

    Directory of Open Access Journals (Sweden)

    Kathleen H Wood

    2016-05-01

    Full Text Available DNA methylation is an epigenetic mark that is essential for many biological processes and is linked to diseases such as cancer. Methylation is usually associated with transcriptional silencing, but new research has challenged this model. Both transcriptional activation and repression have recently been found to be associated with DNA methylation in a context-specific manner. How DNA methylation patterns are interpreted into different functional output remains poorly understood. One mechanism involves the protein ‘readers’ of methylation, which includes the methyl-CpG binding domain (MBD family of proteins. This review examines the molecular and biological functions of MBD2, which binds to CpG methylation and is an integral part of the nucleosome remodeling and histone deacetylation (NuRD complex. MBD2 has been linked to immune system function and tumorigenesis, yet little is known about its functions in vivo. Recent studies have found the MBD2 protein is ubiquitously expressed, with relatively high levels in the lung, liver and colon. Mbd2 null mice surprisingly show relatively mild phenotypes compared to mice with loss of function of other MBD proteins. This evidence has previously been interpreted as functional redundancy between the MBD proteins. Here we examine and contextualize research that suggests MBD2 has unique properties and functions among the MBD proteins. These functions translate to recently described roles in the development and differentiation of multiple cell lineages, including pluripotent stem cells and various cell types of the immune system, as well as in tumorigenesis. We also consider possible models for the dynamic interactions between MBD2 and NuRD in different tissues in vivo. The functions of MBD2 may have direct therapeutic implications for several areas of human disease, including autoimmune conditions and cancer, in addition to providing insights into the actions of NuRD and chromatin regulation.

  4. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

    Directory of Open Access Journals (Sweden)

    Anders Waldenström

    Full Text Available BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

  5. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

    Science.gov (United States)

    Waldenström, Anders; Gennebäck, Nina; Hellman, Urban; Ronquist, Gunnar

    2012-01-01

    Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

  6. The Quantitative and Qualitative Analysis of Cohorts' Early Enrollment in Physics: concurrent with enrollment in mathematics, biology and chemistry

    Science.gov (United States)

    Lynch, Robert Bruce Rodes

    Cohorts of 48 entering biological science majors was recruited in the fall of 2007 and again in 2008 and 2009 for the Interdisciplinary Science Experience (ISE). These ISE students enrolled in their own sections of standard courses of physics, chemistry, and biology. In these courses average ISE student out-performed their non-cohort peers by up to a full letter grade. A qualitative analysis of ISE student interviews illuminates the student experience and shows how the ISE students perceived themselves to be different than their non-cohort peers. Quantitative modeling of student performance shows that higher grades are correlated with multiple factors. These factors includes admissions characteristics such as high school GPA, and SAT scores, as well as demographic information. These trends support and elaborate on the selection narratives told by participants. Additionally the quantitative model found that higher student performance is predicted by structural aspects of the ISE program, specifically the timing of course, enrolling as a freshmen in many of their courses, and the sequencing of physics and chemistry courses. There is a statistically significant benefit to student performance in general and organic chemistry courses associated with completing the first quarter of the Physics for Bio-Science majors prior to enrollment. Further the combination of quantitative and qualitative data suggest that there is a epistemological transfer of problem solving skills and outlook from the physics to the chemistry courses.

  7. Novel Uses of In Vitro Data to Develop Quantitative Biological Activity Relationship Models for in Vivo Carcinogenicity Prediction.

    Science.gov (United States)

    Pradeep, Prachi; Povinelli, Richard J; Merrill, Stephen J; Bozdag, Serdar; Sem, Daniel S

    2015-04-01

    The availability of large in vitro datasets enables better insight into the mode of action of chemicals and better identification of potential mechanism(s) of toxicity. Several studies have shown that not all in vitro assays can contribute as equal predictors of in vivo carcinogenicity for development of hybrid Quantitative Structure Activity Relationship (QSAR) models. We propose two novel approaches for the use of mechanistically relevant in vitro assay data in the identification of relevant biological descriptors and development of Quantitative Biological Activity Relationship (QBAR) models for carcinogenicity prediction. We demonstrate that in vitro assay data can be used to develop QBAR models for in vivo carcinogenicity prediction via two case studies corroborated with firm scientific rationale. The case studies demonstrate the similarities between QBAR and QSAR modeling in: (i) the selection of relevant descriptors to be used in the machine learning algorithm, and (ii) the development of a computational model that maps chemical or biological descriptors to a toxic endpoint. The results of both the case studies show: (i) improved accuracy and sensitivity which is especially desirable under regulatory requirements, and (ii) overall adherence with the OECD/REACH guidelines. Such mechanism based models can be used along with QSAR models for prediction of mechanistically complex toxic endpoints. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    Science.gov (United States)

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Photoexcited triplet state provides a quantitative measure of intercalating drug-DNA binding energies

    Science.gov (United States)

    Maki, August H.; Alfredson, T. V.; Waring, M. J.

    1992-04-01

    A linear correlation between spectroscopic and thermodynamic properties of systems is rarely encountered. In triplet state ODMR studies of various DNA complexes of echinomycin, a quinoxaline-containing cyclic depsipeptide bis-intercalating antibiotic, and its biosynthesized quinoline analogs, such correlations are observed. The zero field splitting D-parameter of the intercalated quinoxaline or quinoline residue varies linearly with the free energy of drug-DNA complexing. From previous work, the DNA sequence specificity of echinomycin analogs is known to be influenced by the identity of the intercalating residue (e.g., quinoxaline vs. quinoline). The present results strongly suggest that the DNA sequence-specificity of these drugs is controlled largely by the intercalated residue, and that the energetics of the peptide- DNA interaction, although considerable, are relatively sequence independent. These conclusions run counter to the generally accepted idea that DNA recognition by sequence- seeking proteins is controlled by specific hydrogen bonding interactions. The high degree of N-methylation of the echinomycin peptide portion severely restricts these interactions, however. A simple theoretical model is presented to support the experimentally observed linear correlation between (Delta) D and (Delta) G.

  10. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    Science.gov (United States)

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

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

    Science.gov (United States)

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

    2004-03-01

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

  12. Quantitative terahertz time-domain spectroscopy and analysis in chemistry and biology

    DEFF Research Database (Denmark)

    Jepsen, Peter Uhd

    2005-01-01

    I will describe how Terahertz Time-Domain Spectroscopy (THz-TDS) can be used for quantitative, broadband spectroscopy in the far-infrared spectral region. Thz-TDS is sensitive to long-range, non-covalent interactions in the condensed phase, for instance intermolecular hydrogen bonding in molecular...

  13. Analytical and Biological Variables Influencing Quantitative Hepatitis C Virus (HCV) Measurement in HIV-HCV Coinfection

    OpenAIRE

    Curtis Cooper; Paul MacPherson; William Cameron

    2006-01-01

    The present review considers issues pertaining to the precision and variability of quantitative hepatitis C virus (HCV) measurement in general, outlines the characteristics of HCV RNA in HIV-HCV coinfection and evaluates those factors which may affect this measure. The clinical relevance of accurate HCV measurement in HIV-HCV coinfection is discussed.

  14. Quantitative analysis of biological tissues using Fourier transform-second-harmonic generation imaging

    Science.gov (United States)

    Ambekar Ramachandra Rao, Raghu; Mehta, Monal R.; Toussaint, Kimani C., Jr.

    2010-02-01

    We demonstrate the use of Fourier transform-second-harmonic generation (FT-SHG) imaging of collagen fibers as a means of performing quantitative analysis of obtained images of selected spatial regions in porcine trachea, ear, and cornea. Two quantitative markers, preferred orientation and maximum spatial frequency are proposed for differentiating structural information between various spatial regions of interest in the specimens. The ear shows consistent maximum spatial frequency and orientation as also observed in its real-space image. However, there are observable changes in the orientation and minimum feature size of fibers in the trachea indicating a more random organization. Finally, the analysis is applied to a 3D image stack of the cornea. It is shown that the standard deviation of the orientation is sensitive to the randomness in fiber orientation. Regions with variations in the maximum spatial frequency, but with relatively constant orientation, suggest that maximum spatial frequency is useful as an independent quantitative marker. We emphasize that FT-SHG is a simple, yet powerful, tool for extracting information from images that is not obvious in real space. This technique can be used as a quantitative biomarker to assess the structure of collagen fibers that may change due to damage from disease or physical injury.

  15. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  16. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

    Science.gov (United States)

    Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

    2015-07-20

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

  17. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    Science.gov (United States)

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

  18. Quantitative, high-resolution proteomics for data-driven systems biology

    DEFF Research Database (Denmark)

    Cox, J.; Mann, M.

    2011-01-01

    Systems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem...... primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics...... data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far. © 2011 by Annual Reviews. All rights reserved....

  19. Systems Biology of Cancer: A Challenging Expedition for Clinical and Quantitative Biologists

    Directory of Open Access Journals (Sweden)

    Ilya eKorsunsky

    2014-08-01

    Full Text Available A systems-biology approach to complex disease (such as cancer is now complementing traditional experience-based approaches, which have typically been invasive and expensive. The rapid progress in biomedical knowledge is enabling the targeting of disease with therapies that are precise, proactive, preventive and personalized. In this paper, we summarize and classify models of systems biology and model-checking tools which have been used to great success in computational biology and related fields. We demonstrate how these models and tools have been used to study some of the twelve biochemical pathways implicated in but not unique to pancreatic cancer, and conclude that the resulting mechanistic models will need to be further enhanced by various abstraction techniques to interpret phenomenological models of cancer progression.

  20. Systems biology of cancer: a challenging expedition for clinical and quantitative biologists.

    Science.gov (United States)

    Korsunsky, Ilya; McGovern, Kathleen; LaGatta, Tom; Olde Loohuis, Loes; Grosso-Applewhite, Terri; Griffeth, Nancy; Mishra, Bud

    2014-01-01

    A systems-biology approach to complex disease (such as cancer) is now complementing traditional experience-based approaches, which have typically been invasive and expensive. The rapid progress in biomedical knowledge is enabling the targeting of disease with therapies that are precise, proactive, preventive, and personalized. In this paper, we summarize and classify models of systems biology and model checking tools, which have been used to great success in computational biology and related fields. We demonstrate how these models and tools have been used to study some of the twelve biochemical pathways implicated in but not unique to pancreatic cancer, and conclude that the resulting mechanistic models will need to be further enhanced by various abstraction techniques to interpret phenomenological models of cancer progression.

  1. Quantitative criteria for improving performance of buccal DNA for high-throughput genetic analysis

    Directory of Open Access Journals (Sweden)

    Woo Jessica G

    2012-08-01

    Full Text Available Abstract Background DNA from buccal brush samples is being used for high-throughput analyses in a variety of applications, but the impact of sample type on genotyping success and downstream statistical analysis remains unclear. The objective of the current study was to determine laboratory predictors of genotyping failure among buccal DNA samples, and to evaluate the successfully genotyped results with respect to analytic quality control metrics. Sample and genotyping characteristics were compared between buccal and blood samples collected in the population-based Genetic and Environmental Risk Factors for Hemorrhagic Stroke (GERFHS study (https://gerfhs.phs.wfubmc.edu/public/index.cfm. Results Seven-hundred eight (708 buccal and 142 blood DNA samples were analyzed for laboratory-based and analysis metrics. Overall genotyping failure rates were not statistically different between buccal (11.3% and blood (7.0%, p = 0.18 samples; however, both the Contrast Quality Control (cQC rate and the dynamic model (DM call rates were lower among buccal DNA samples (p  Conclusions We identified a buccal sample characteristic, a ratio of ds/total DNA

  2. Cold Spring Harbor symposia on quantitative biology. Volume 48. Molecular neurobiology

    Energy Technology Data Exchange (ETDEWEB)

    1983-01-01

    A new mood exists among those molecular biologists who gaze upwards toward the brain. By using recombinant DNA, monoclonal antibodies, and the facts of receptor biochemistry to the fullest, we should have a fighting chance to understand the uniqueness of nerve cells and the ways they come together to form functional networks. To mark this new mood, the 48th Symposium on Molecular Neurobiology was held. These proceedings contain 89 separate papers divided into subject areas including acetylcholine receptor and its channel, sodium channel, calcium and potassium channels, studies on neuronal proteins with recombinant DNA techniques, molecular aspects of neuropeptides, genetic analysis of the nervous system, neuronal surface molecules, the regulation of axon organization, synaptic organization and function, the cellular organization of neurons, behavior, and aspects of vision. Individual papers were also abstracted and indexed for the Energy Data Base.

  3. From classical genetics to quantitative genetics to systems biology: modeling epistasis.

    Directory of Open Access Journals (Sweden)

    David L Aylor

    2008-03-01

    Full Text Available Gene expression data has been used in lieu of phenotype in both classical and quantitative genetic settings. These two disciplines have separate approaches to measuring and interpreting epistasis, which is the interaction between alleles at different loci. We propose a framework for estimating and interpreting epistasis from a classical experiment that combines the strengths of each approach. A regression analysis step accommodates the quantitative nature of expression measurements by estimating the effect of gene deletions plus any interaction. Effects are selected by significance such that a reduced model describes each expression trait. We show how the resulting models correspond to specific hierarchical relationships between two regulator genes and a target gene. These relationships are the basic units of genetic pathways and genomic system diagrams. Our approach can be extended to analyze data from a variety of experiments, multiple loci, and multiple environments.

  4. Quantitative evaluation of p53 as a new indicator of DNA damage in human spermatozoa

    Directory of Open Access Journals (Sweden)

    Salvatore Raimondo

    2014-01-01

    The aim of this study was to assess if a p53 ELISA assay could be a new indicator of DNA damage in human spermatozoa. Materials and Methods: 103 human semen samples were evaluated using both Acridine Orange test and p53 ELISA and results were compared. Results: A clear correlation between the values measured by two methods was obtained. Conclusions: If this hypothesis will be confirmed by further studies, the p53 ELISA assay could become a new and more precise indicator of DNA damage in human spermatozoa.

  5. An Assessment of the Quantitative Skills of Students Taking Introductory College Biology Courses.

    Science.gov (United States)

    Marsh, Jeffrey Flake; Anderson, Norman D.

    The mathematical skills possessed by students taking introductory biology courses were investigated. A list of 23 mathematical competencies was identified as part of the development of a 46-item multiple-choice test to measure the extent to which students possessed these competencies. The Biomathematics Skills Test (BST) was administered to…

  6. Plant molecular biology and biotechnology research in the post-recombinant DNA era.

    Science.gov (United States)

    Tyagi, Akhilesh K; Khurana, Jitendra P

    2003-01-01

    After the beginning of the recombinant DNA era in the mid-1970s, researchers in India started to make use of the new technology to understand the structure of plant genes and regulation of their expression. The outcome started to appear in print in early the 1980s and genes for histones, tubulin, photosynthetic membrane proteins, phototransduction components, organelles and those regulated differentially by developmental and extrinsic signals were sequenced and characterized. Some genes of biotechnological importance like those encoding an interesting seed protein and the enzyme glyoxalase were also isolated. While work on the characterization of genome structure and organization was started quite early, it remained largely focused on the identification of DNA markers and genetic variability. In this context, the work on mustard, rice and wheat is worth mentioning. In the year 2000, India became a member of the international consortium to sequence entire rice genome. Several laboratories have also given attention to regulated expression of plastid and nuclear genes as well as to isolate target-specific promoters or design promoters with improved potential. Simultaneously, transgenic systems for crops like mustard, rice, wheat, cotton, legumes and several vegetables have been established. More recently, genes of agronomic importance like those for insect resistance, abiotic stress tolerance, nutritional improvement and male sterility, isolated in India or abroad, have been utilized for raising transgenics for crop improvement. Some of these transgenics have already shown their potential in containment facility or limited field trials conducted under the stipulated guidelines. Plant molecular biology and biotechnology are thus clearly poised to make an impact on research in basic biology and agriculture in the near future.

  7. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  8. Quantitative studies of DNA methylation and gene expression in neuropsychiatric traits

    NARCIS (Netherlands)

    van Eijk, K.R.

    2014-01-01

    Research has shown that besides genes and environment, epigenetic factors are also playing a role in the development of traits and diseases. Epigenetic changes do not affect the underlying DNA sequence, but affect its structure and is thought to play a major role in regulation of gene expression. DN

  9. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    Science.gov (United States)

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  10. The near-quantitative sampling of genomic DNA from various food-borne Eubacteria

    National Research Council Canada - National Science Library

    Irwin, Peter; Nguyen, Ly; He, Yiping; Paoli, George; Gehring, Andrew; Chen, Chin-Yi

    2014-01-01

    .... In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70 × 10(6) (±9.05%), 4.77 × 10(8) (±31.0%), and 5.93 × 10(8) (±4.69...

  11. Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Nataڑa Vasiljeviš

    2011-01-01

    Full Text Available Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa. We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH samples using the pyrosequencing (PSQ method to identify genes with diagnostic and prognostic potential.

  12. Quantitative analysis of the ion-dependent folding stability of DNA triplexes.

    Science.gov (United States)

    Chen, Gengsheng; Chen, Shi-Jie

    2011-12-01

    A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.

  13. Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples.

    Science.gov (United States)

    Zhang, Wei; Wu, Meng; Menesale, Emily; Lu, Tongjun; Magliola, Aeona; Bergelson, Svetlana

    2014-11-01

    Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.

  14. Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.

    Science.gov (United States)

    Kraytsberg, Yevgenya; Bodyak, Natalya; Myerow, Susan; Nicholas, Alexander; Ebralidze, Konstantin; Khrapko, Konstantin

    2009-01-01

    Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).

  15. Comparison of Three Different Methods for Quantitative Analysis of DNA Methylation%3种 DNA 甲基化定量分析方法的比较

    Institute of Scientific and Technical Information of China (English)

    路兆宁; 周在威; 马晴雯; 颜景斌

    2015-01-01

    Objective To compare the advantages and disadvantages of different methods for quantitative analysis of locus-specific DNA methylation. Methods Three different methods inclu-ding bisulfite pyrosequencing, bisulfite sequencing of clones and methylated DNA immunoprecipita-tion (MeDIP) -chip were used to identify the DNA methylation level at CpG island of PRDM8 in-tron 7 in 7 Down syndrome (DS) and 5 normal samples. To choose the suitable methods for quantita-tive analysis of locus-specific DNA methylation, the accuracy, repeatability and resolution of these methods were compared. Results DNA methylation was identified at single-base resolution by bisul-fite pyrosequencing with high accuracy and good reproducibility. More importantly, the variation tendency of methylation levels at different CpG sites was found with bisulfite pyrosequencing. However, these advantages were not revealed with bisulfite sequencing of clones, though it was a useful method for the detection of methylation at single-nucleotide resolution. In addi-tion, relative enrichment of methylated DNA was obtained by MeDIP-chip through the signals of two probes corresponding to the detection sequence. Nevertheless, it was not suitable for detection of methylation at single-nucleotide resolution. In this study, hypermethylation was observed at CpG is-land of PRDM8 intron 7 in DS, although some differences existed in three methods. Conclusion Bisulfite pyrosequencing is a suitable method for quantitative analysis of locus-specific DNA methyla-tion at single-nucleotide resolution with some prominent characteristics, such as higher accuracy, excellent reproducibility, lower costs and more convenient operation.%目的:比较特定基因区域 DNA 甲基化检测方法的优缺点。方法采用焦磷酸测序、克隆测序和DNA 甲基化芯片等3种 DNA 甲基化检测方法,对7例唐氏综合征和5例正常对照样本的 PRDM8内含子7 CpG 岛中的部分序列进行甲基化检测。从准确

  16. 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

  17. 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; De Jonghe, Joachim; 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.

  18. Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

    Science.gov (United States)

    Pritzkow, Sandra; Wagenführ, Katja; Daus, Martin L.; Boerner, Susann; Lemmer, Karin; Thomzig, Achim; Mielke, Martin; Beekes, Michael

    2011-01-01

    Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity

  19. Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection.

    Directory of Open Access Journals (Sweden)

    Sandra Pritzkow

    Full Text Available Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤10(1- to ≥10(5.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological

  20. Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection.

    Science.gov (United States)

    Pritzkow, Sandra; Wagenführ, Katja; Daus, Martin L; Boerner, Susann; Lemmer, Karin; Thomzig, Achim; Mielke, Martin; Beekes, Michael

    2011-01-01

    Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤10(1)- to ≥10(5.5)-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie

  1. A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    Jason R. Schwartz; Purvaba J. Sarvaiya; Lily E. Leiva; Maria C. Velez; Tammuella C. Singleton; Lolie C. Yu; Wayne V. Vedeckis

    2012-01-01

    Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript (exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

  2. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA.

    Science.gov (United States)

    Lorenzi, Julio C C; Cohen, Yehuda Z; Cohn, Lillian B; Kreider, Edward F; Barton, John P; Learn, Gerald H; Oliveira, Thiago; Lavine, Christy L; Horwitz, Joshua A; Settler, Allison; Jankovic, Mila; Seaman, Michael S; Chakraborty, Arup K; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-12-06

    HIV-1-infected individuals harbor a latent reservoir of infected CD4(+) T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

  3. Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wu Dan; Chi Hongbin; Shao Minjie; Wu Yao; Jin Hongyan; Wu Baiyan; Qiao Jie

    2014-01-01

    Backgroud Amniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval,CI:77%-98%) and the specificity was 100% (26/26; CI:88%-100%).The determination rate of the origin of the extra chromosome was 69%.The sensitivity and the specificity of the assay in the euploid were 100% (27/27).Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

  4. In-silico screening for DNA-dependent protein kinase (DNA-PK) inhibitors: Combined homology modeling, docking, molecular dynamic study followed by biological investigation.

    Science.gov (United States)

    Tarazi, Hamadeh; Saleh, Ekram; El-Awady, Raafat

    2016-10-01

    DNA-dependent protein kinase (DNA-PK) is a key enzyme in non-homologous DNA end joining (NHEJ) repair pathway. The targeted inhibition of such enzyme would furnish a valuable option for cancer treatment. In this study we report the development of validation of enzyme homology model, and the subsequent use of this model to perform docking-based virtual screening against a database of FDA-approved drugs. The nominated highest ranking hits (Praziquantel and Dutasteride) were subjected to biological investigation. Additionally, molecular dynamic study was carried-out for binding mode exploration. Results of the biological evaluation revealed that both compounds inhibit the DNA-PK enzymatic activity at relatively high concentration levels with an IC50 of 17.3μM for praziquantel and >20μM for dutasteride. Furthermore, both agents enhanced the anti-proliferative effects of doxorubicin and cisplatin on breast cancer (MCF7) and lung cancer (A549) cell lines. This result indicates that these two hits are good candidate as DNA-PK inhibitors and worth further structural modifications to enhance their enzyme inhibitory effects. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Near-quantitative extraction of genomic DNA from various food-borne eubacteria

    Science.gov (United States)

    In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70E6 (± 9.05%), 4.77E8 (± 31.0%), and 5.93E8 (± 4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt), Shigella sonnei (Ss), and Esch...

  6. A quantitative PCR method to quantify ruminant DNA in porcine crude heparin

    OpenAIRE

    Concannon, Sean P.; Wimberley, P. Brett; Workman, Wesley E.

    2010-01-01

    Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quant...

  7. Accurate quantitation of allele-specific expression patterns by analysis of DNA melting

    OpenAIRE

    Jeong, Sangkyun; Hahn, Yoonsoo; Rong, Qi; Pfeifer, Karl

    2007-01-01

    Epigenetic and genetic mechanisms can result in large differences in expression levels of the two alleles in a diploid organism. Furthermore, these differences may be critical to phenotypic variations among individuals. In this study, we present a novel procedure and algorithm to precisely and accurately quantitate the relative expression of each allele. This method uses the differential melting properties of DNAs differing at even a single base pair. By referring to the melting characteristi...

  8. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  9. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology.

    Science.gov (United States)

    Li, Jian-Feng; Li, Li; Sheen, Jen

    2010-01-14

    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  10. Effect of Co-Existing Biologically Relevant Molecules and Ions on DNA Photocleavage Caused by Pyrene and its Derivatives

    Directory of Open Access Journals (Sweden)

    Hongtao Yu

    2005-04-01

    Full Text Available Inorganic ions, coenzymes, amino acids, and saccharides could co-exist with toxic environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs, in the cell. The presence of these co-existing chemicals can modulate the toxicity of the PAHs. One of the genotoxic effects by PAHs is light-induced cleavage, or photocleavage, of DNA. The effect of inorganic ions I-, Na+, Ca2+, Mg2+, Fe3+, Mn2+, Cu2+, and Zn2+ and biological molecules riboflavin, histidine, mannitol, nicotinamide adenine dinucleotide (NAD, glutathione, and glutamic acid on the DNA photocleavage by pyrene, 1-hydroxypyrene (1-HP, and 1-aminopyrene (1-AP, is studied. The non-transition metal ions Na+, Ca2+, and Mg2+, usually have very little inhibitory effects, while the transition metal ions Fe3+, Cu2+, and Zn2+ enhance, Mn2+ inhibits the DNA photocleavage. The effect by biological molecules is complex, depending on the photochemical reaction mechanisms of the compounds tested (1-AP, 1-HP and pyrene and on the chemical nature of the added biological molecules. Riboflavin, histidine, and mannitol enhance DNA photocleavage by all three compounds, except that mannitol has no effect on the photocleavage of DNA by pyrene. Glutathione inhibits the DNA photocleavage by 1-AP and 1-HP, but has no effect on that by pyrene. NAD enhances the DNA photocleavage by 1-AP, but has no effect on that by 1-HP and pyrene. Glutamic acid enhances the DNA photocleavage by 1-AP and pyrene, but inhibits that by 1-HP. These results show that the co-existing chemicals may have a profound effect on the toxicity of PAHs, or possibly on the toxicity of many other chemicals. Therefore, if one studies the toxic effects of PAHs or other toxic chemicals, the effect of the co-existing chemicals or ions needs to be considered.

  11. Informatics and Physics Models of Recognitions of DNA Replication and Their Biological Applications

    Directory of Open Access Journals (Sweden)

    Kang Cheng

    2006-01-01

    Full Text Available We initially propose concepts of Informative Intensity, Informative Response Intensity and Informative Flux, with different expressions. In a special expression of electrostatics, we describe Informative Intensity, Informative Response Intensity and Informative Flux in terms of electric field intensity, electric field force and electric field flux respectively. In a special expression of quantum mechanics, we originally propose a concept of Probability Flux. Then we present Informative Intensity in terms of a wave function of Schrödinger equation and Informative Response Intensity in terms of an interactive force between or among objects, Informative Flux in terms of a probability flux. Based on these concepts, we develop our Informatics and physics models of recognitions between a DNA polymerase and an initiation site and of pairing deoxyribonucleotides, for a natural DNA replication, beyond lengths of chemical bounds and half quantitatively explain a probability of the wrong paring. We originally hypothesize the information is, stored in structured charges or masses, transmitted with Informative Intensity in a media and recognized with an Informative Response Intensity by other structured charges or masses. We further generalize, Informative Intensity as multiple layers of fields, potentials or waves, Informative Response Intensity as multiple layers of forces and Informative Flux as an integration of Informative Intensity. We also initially define Transverse Information: Informative Intensity is perpendicular to the direction of information propagation, e.g. electromagnetic wave or magnetic field; and Longitudinal Information: Informative Intensity is parallel or antiparalell to the direction of information propagation, e.g. electric field or gravitational field.

  12. Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

    Directory of Open Access Journals (Sweden)

    Prakash Neeraj

    2010-11-01

    Full Text Available Abstract Background- Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR, apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology. Results- The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001, DCR1 (P = 0.00001, DCR2 (P = 0.0000000005 and BRCA2 (P = 0.007 and hypomethylation of DR4 (P = 0.011 in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047 and DNA damage repair potential (P = 0.004 in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors. Conclusion- Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing

  13. Mapping of thermal injury in biologic tissues using quantitative pathologic techniques

    Science.gov (United States)

    Thomsen, Sharon L.

    1999-05-01

    Qualitative and quantitative pathologic techniques can be used for (1) mapping of thermal injury, (2) comparisons lesion sizes and configurations for different instruments or heating sources and (3) comparisons of treatment effects. Concentric zones of thermal damage form around a single volume heat source. The boundaries between some of these zones are distinct and measurable. Depending on the energy deposition, heating times and tissue type, the zones can include the following beginning at the hotter center and progressing to the cooler periphery: (1) tissue ablation, (2) carbonization, (3) tissue water vaporization, (4) structural protein denaturation (thermal coagulation), (5) vital enzyme protein denaturation, (6) cell membrane disruption, (7) hemorrhage, hemostasis and hyperhemia, (8) tissue necrosis and (9) wound organization and healing.

  14. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

    Science.gov (United States)

    Purcell, Maureen K.; Kurath, Gael; Garver, Kyle A.; Herwig, Russell P.; Winton, James R.

    2004-01-01

    Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

  15. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    Science.gov (United States)

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  16. Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

    Directory of Open Access Journals (Sweden)

    Adam G. Marsh

    2016-11-01

    Full Text Available Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS data using methyl-sensitive restriction endonucleases (MSRE. An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1,008 statistically different CpG sites (p textless 0.0025, after false discovery rate correction. A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations. Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

  17. QUANTITATIVE ESTIMATION OF DNA ISOLATED FROM VARIOUS PARTS OF ANNONA SQUAMOSA

    Directory of Open Access Journals (Sweden)

    Soni Himesh

    2011-12-01

    Full Text Available Plants have been one of the important sources of medicines since the beginning of human civilization. There is a growing demand for plant based medicines, health products, pharmaceuticals, food supplements, cosmetics etc. Annona squamosa Linn is a multipurpose tree with edible fruits & is a source one of the medicinal & industrial products. Annona squamosa Linn is used as an antioxidant, antidiabetics, hepatoprotective, cytotoxicactivity, genetoxicity, antitumor activity, antilice agent. It is related to contain alkaloids, flavonoids, carbohydrates, fixed oils, tannins & phenolic. Genetic variation is essential for long term survival of species and it is a critical feature in conservation. For efficient conservation and management, the genetic composition of the species in different geographic locations needs to be assessed. Plants are attracting more attention among contemporary pharmacy scientists because some human diseases resulting from antibiotic resistance have gained worldwide concern. A number of methods are available and are being developed for the isolation of nucleic acids from plants. The different parts of Annona squamosa were studied for their nucleic acid content by using spectrophotometric analysis. In order to measure DNA content of the Leaves,friuts and stems of Annona squamosa, Spectrophotometry serves various advantages i.e. non-destructive and allows the sample to be recovered for further analysis or manipulation. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. This article deals with modern approaches to develop a simple, efficient, reliable and cost-effective method for isolation, separation and estimation of total genomic DNA from various parts of the same species.

  18. Biological Matrix Effects in Quantitative Tandem Mass Spectrometry-Based Analytical Methods: Advancing Biomonitoring

    Science.gov (United States)

    Panuwet, Parinya; Hunter, Ronald E.; D’Souza, Priya E.; Chen, Xianyu; Radford, Samantha A.; Cohen, Jordan R.; Marder, M. Elizabeth; Kartavenka, Kostya; Ryan, P. Barry; Barr, Dana Boyd

    2015-01-01

    The ability to quantify levels of target analytes in biological samples accurately and precisely, in biomonitoring, involves the use of highly sensitive and selective instrumentation such as tandem mass spectrometers and a thorough understanding of highly variable matrix effects. Typically, matrix effects are caused by co-eluting matrix components that alter the ionization of target analytes as well as the chromatographic response of target analytes, leading to reduced or increased sensitivity of the analysis. Thus, before the desired accuracy and precision standards of laboratory data are achieved, these effects must be characterized and controlled. Here we present our review and observations of matrix effects encountered during the validation and implementation of tandem mass spectrometry-based analytical methods. We also provide systematic, comprehensive laboratory strategies needed to control challenges posed by matrix effects in order to ensure delivery of the most accurate data for biomonitoring studies assessing exposure to environmental toxicants. PMID:25562585

  19. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Ogrinc, Nina [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); LOTRIČ Metrology, Selca 163, SI-4227 Selca (Slovenia); Pelicon, Primož, E-mail: primoz.pelicon@ijs.si [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Vavpetič, Primož; Kelemen, Mitja; Grlj, Nataša; Jeromel, Luka [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Tomić, Sergej [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Čolić, Miodrag [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Medical Faculty, University of Niš, Boulevard of Dr. Zoran Djindjić 81, 18000 Niš (Serbia); Beran, Alfred [Dipartimento di Oceanografia Biologica, Istituto Nazionale di Oceanografia e Geofisica Sperimentale, Via Auguste Piccard 54, 34151 Trieste (Italy)

    2013-07-01

    Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10{sup −12} g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

  20. Colour patterns do not diagnose species: quantitative evaluation of a DNA barcoded cryptic bumblebee complex.

    Directory of Open Access Journals (Sweden)

    James C Carolan

    Full Text Available Cryptic diversity within bumblebees (Bombus has the potential to undermine crucial conservation efforts designed to reverse the observed decline in many bumblebee species worldwide. Central to such efforts is the ability to correctly recognise and diagnose species. The B. lucorum complex (Bombus lucorum, B. cryptarum and B. magnus comprises one of the most abundant and important group of wild plant and crop pollinators in northern Europe. Although the workers of these species are notoriously difficult to diagnose morphologically, it has been claimed that queens are readily diagnosable from morphological characters. Here we assess the value of colour-pattern characters in species identification of DNA-barcoded queens from the B. lucorum complex. Three distinct molecular operational taxonomic units were identified each representing one species. However, no uniquely diagnostic colour-pattern character state was found for any of these three molecular units and most colour-pattern characters showed continuous variation among the units. All characters previously deemed to be unique and diagnostic for one species were displayed by specimens molecularly identified as a different species. These results presented here raise questions on the reliability of species determinations in previous studies and highlights the benefits of implementing DNA barcoding prior to ecological, taxonomic and conservation studies of these important key pollinators.

  1. Rings and ladders in biology - fast ab initio simulations of polypeptides and DNA.

    Science.gov (United States)

    Lewis, James P.

    1996-03-01

    Throughout the years, developments of first principles methods have allowed a theoretical investigation of a wide variety of materials from semiconductors to zeolites. However, ab initio methods have not been widespread in the area of large biological systems. Several recent advances in theoretical techniques have prompted us to examine the possibility of simulating large biological systems. Linear scaling methods have been developed to avoid the N^3 computational roadblock due to matrix diagonalization, and a hydrogen-bonding model has been developed to correctly model weak intermolecular interactions within a tight-binding like local orbital framework.(J. Ortega, J. P. Lewis, O. F. Sankey Phys. Rev. B. 50), 10516 (1994); J. P. Lewis and O. F. Sankey, Biophys. J. 69, 1068 (1995). With these developments, a simulation of a dehydrated 10 basepair poly(dG) -- poly(dC) segment of DNA will be described. Results for the electronic structure of this relaxed structure will be discussed. In addition, a simulation of this relaxed structure, involving 1932 steps, was performed to determine the dynamical matrix. The corresponding vibrational spectrum was found and trends will be compared with experimental work.(Work done in collaboration with Otto F. Sankey and Pablo Ordejón) In addition, theoretical results on the energetics, electronic, vibrational and elastic properties of cyclic peptide systems cyclo[(D-Ala-Glu-D-Ala-Gln)_m], where m=1-4, will be presented. Experimentally, these cyclic peptide nanotubes have been shown to be excellent for transporting of ions and glucose across membranes, the attempt to simulate the placement of a dopant into the nanotube structure and the effects on the electronic structure will be discussed.(Work done in collaboration with Otto F. Sankey and Norma H. Pawley)

  2. Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

    Institute of Scientific and Technical Information of China (English)

    孙跃明; 王学浩; 杜竞辉

    2001-01-01

    Objective To obtain IL-2 gene transfected human liver cancer cells and study IL-2 expression and its biologic activity in vivo. Methods Human liver cancer cells SMMC-7721 were cocultured with recombinant retroviral vector LNC-IL-2,and screening was performed in G418 medium.The exogenous IL-2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR-PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion The IL-2 cDNA was successfully integrated into SMMC-7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo.

  3. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

    Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  4. Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

    Directory of Open Access Journals (Sweden)

    Burgess J Grant

    2009-06-01

    Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

  5. Effects of Single and Combined Application of Organic and Biological Fertilizers on Quantitative and Qualitative Yield of Anisum (Pimpinella anisum

    Directory of Open Access Journals (Sweden)

    N Kamayestani

    2015-07-01

    Full Text Available In order to study the effects of single and combined applications of biofertilazer and organic fertilizers on quantitative and qualitative characteristics of anisum (Pimpinella anisum, an experiment was conducted based on a Randomized Complete Block Design with three replications and fifteen treatments at Research Station, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran, in 2011 year. Treatments were: (1 mycorrhiza (Glomus intraradices, (2 mycorrhiza + cow manure, (3 mycorrhiza + vermicompost, (4 mycorrhiza+ compost, (5 mycorrhiza + chemical fertilizer, (6 biosulfur (Thiobacillus sp. + Bentonite, (7 biosulfur + chemical fertilizer, (8 biosulfur + cow manure, (9 biosulfur + vermicompost, (10 biosulfur+compost,11 (cow manure, (12 vermicompost, (13 chemical fertilizer (NPK, (14compost and (15 control. The results showed that application of fertilizer treatments had significant effect on most characteristics of anisum. The highest number of seed per umbelet (7.24, economic yield (1263.4kg/ha were obtained fram biosulfur treatment. The highest dry matter yield (4504.1 kg/ha resulted from combined application of biosulfur + chemical fertilizer and the highest harvest index (25.97% observed in biosulfur+cow manure. The combined application of mycorrhiza affected some qualification traits, as the highest number of umbel per plant (65.7, 1000 seed-weight (3.24 g and essential oil percentage (5.3% resulted from combined application of mycorrhiza+chemical fertilizer. In general, it can be concluded that application of organic and biological fertilizer particularly mycorrhiza and biosulfur had a significant effect on improving of quantitative and qualitative characteristics of anisum. Furthermore, the combined application of organic and biological fertilizer had higher positive effects than their single application.

  6. A bench-top K X-ray fluorescence system for quantitative measurement of gold nanoparticles for biological sample diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Ricketts, K., E-mail: k.ricketts@ucl.ac.uk [Division of Surgery and Interventional Sciences, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF (United Kingdom); Guazzoni, C.; Castoldi, A. [Dipartimento di Elettronica, Informazione e Bioingegneria Politecnico di Milano and INFN, Sezione di Milano P.za Leonardo da Vinci, 32-20133 Milano (Italy); Royle, G. [Department of Medical Physics and Bioengineering, University College London, Malet Place Engineering Building, Gower Street, London WC1E 6BT (United Kingdom)

    2016-04-21

    Gold nanoparticles can be targeted to biomarkers to give functional information on a range of tumour characteristics. X-ray fluorescence (XRF) techniques offer potential quantitative measurement of the distribution of such heavy metal nanoparticles. Biologists are developing 3D tissue engineered cellular models on the centimetre scale to optimise targeting techniques of nanoparticles to a range of tumour characteristics. Here we present a high energy bench-top K-X-ray fluorescence system designed for sensitivity to bulk measurement of gold nanoparticle concentration for intended use in such thick biological samples. Previous work has demonstrated use of a L-XRF system in measuring gold concentrations but being a low energy technique it is restricted to thin samples or superficial tumours. The presented system comprised a high purity germanium detector and filtered tungsten X-ray source, capable of quantitative measurement of gold nanoparticle concentration of thicker samples. The developed system achieved a measured detection limit of between 0.2 and 0.6 mgAu/ml, meeting specifications of biologists and being approximately one order of magnitude better than the detection limit of alternative K-XRF nanoparticle detection techniques. The scatter-corrected K-XRF signal of gold was linear with GNP concentrations down to the detection limit, thus demonstrating potential in GNP concentration quantification. The K-XRF system demonstrated between 5 and 9 times less sensitivity than a previous L-XRF bench-top system, due to a fundamental limitation of lower photoelectric interaction probabilities at higher K-edge energies. Importantly, the K-XRF technique is however less affected by overlying thickness, and so offers future potential in interrogating thick biological samples.

  7. A quantitative model of DNA replication in Xenopus embryos: reliable replication despite stochasticity

    Science.gov (United States)

    Cheng-Hsin Yang, Scott; Bechhoefer, John

    2008-03-01

    DNA synthesis in Xenopus frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the replication time and may lead to cell death if replication takes longer than the cell cycle time (˜ 25 min.). Surprisingly, although the typical replication time is about 20 min., in vivo experiments show that replication fails to complete only about 1 in 250 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two mechanisms: the first uses a regular spatial distribution of origins, while the second uses randomly located origins but increases their probability of initiation as the cell cycle proceeds. Here, we show that both mechanisms yield similar end-time distributions, implying that regular origin spacing is not needed for control of replication time. Moreover, we show that the experimentally inferred time-dependent initiation rate satisfies the observed low failure probability and nearly optimizes the use of replicative proteins.

  8. 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.

  9. Host cell proteins in biologics development: Identification, quantitation and risk assessment.

    Science.gov (United States)

    Wang, Xing; Hunter, Alan K; Mozier, Ned M

    2009-06-15

    Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications. 2009 Wiley Periodicals, Inc.

  10. Teacher knowledge and discourse control: Quantitative evidence from novice biology teachers' classrooms

    Science.gov (United States)

    Carlsen, William S.

    This article describes the effects of science teacher subject-matter knowledge on classroom discourse at the level of individual utterances. It details one of three parallel analyses conducted in a year-long study of language in the classrooms of four new biology teachers. The conceptual framework of the study predicts that when teaching unfamiliar subject matter, teachers use a variety of discourse strategies to constrain student talk to a narrowly circumscribed topic domain. This article includes the results of an utterance-by-utterance analysis of teacher and student talk in a 30-lesson sample of science instruction. Data are broken down by classroom activity (e.g., lecture, laboratory, group work) for several measures, including mean duration of utterances, domination of the speaking floor by the teacher, frequency of teacher questioning, cognitive level of teacher questions, and student verbal participation. When teaching unfamiliar topics, the four teachers in this study tended to talk more often and for longer periods of time, ask questions frequently, and rely heavily on low cognitive level questions. The rate of student questions to the teacher varied with classroom activity. In common classroom communicative settings, student questions were less common when the teacher was teaching unfamiliar subject matter. The implications of these findings include a suggestion that teacher knowledge may be an important unconsidered variable in research on the cognitive level of questions and teacher wait-time.

  11. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, R.M.; Nanda, S.; Altom, M.G.

    1984-01-01

    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  12. Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

    Science.gov (United States)

    Zhang, Ying; Huang, Tao; Jorgens, Danielle M; Nickerson, Andrew; Lin, Li-Jung; Pelz, Joshua; Gray, Joe W; López, Claudia S; Nan, Xiaolin

    2017-01-01

    Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

  13. Nucleobase recognition at alkaline pH and apparent pK(a) of single DNA bases immobilised within a biological nanopore

    NARCIS (Netherlands)

    Fransceschini, Lorenzo; Mikhailova, Ellina; Bayley, Hagan; Maglia, Giovanni

    2012-01-01

    The four DNA bases are recognized in immobilized DNA strands at high alkaline pH by nanopore current recordings. Ionic currents through the biological nanopores are also employed to measure the apparent pK(a) values of single nucleobases within the immobilised DNA strands.

  14. DNA methylation-based measures of biological age: meta-analysis predicting time to death

    Science.gov (United States)

    Chen, Brian H.; Marioni, Riccardo E.; Colicino, Elena; Peters, Marjolein J.; Ward-Caviness, Cavin K.; Tsai, Pei-Chien; Roetker, Nicholas S.; Just, Allan C.; Demerath, Ellen W.; Guan, Weihua; Bressler, Jan; Fornage, Myriam; Studenski, Stephanie; Vandiver, Amy R.; Moore, Ann Zenobia; Tanaka, Toshiko; Kiel, Douglas P.; Liang, Liming; Vokonas, Pantel; Schwartz, Joel; Lunetta, Kathryn L.; Murabito, Joanne M.; Bandinelli, Stefania; Hernandez, Dena G.; Melzer, David; Nalls, Michael; Pilling, Luke C.; Price, Timothy R.; Singleton, Andrew B.; Gieger, Christian; Holle, Rolf; Kretschmer, Anja; Kronenberg, Florian; Kunze, Sonja; Linseisen, Jakob; Meisinger, Christine; Rathmann, Wolfgang; Waldenberger, Melanie; Visscher, Peter M.; Shah, Sonia; Wray, Naomi R.; McRae, Allan F.; Franco, Oscar H.; Hofman, Albert; Uitterlinden, André G.; Absher, Devin; Assimes, Themistocles; Levine, Morgan E.; Lu, Ake T.; Tsao, Philip S.; Hou, Lifang; Manson, JoAnn E.; Carty, Cara L.; LaCroix, Andrea Z.; Reiner, Alexander P.; Spector, Tim D.; Feinberg, Andrew P.; Levy, Daniel; Baccarelli, Andrea; van Meurs, Joyce; Bell, Jordana T.; Peters, Annette; Deary, Ian J.; Pankow, James S.; Ferrucci, Luigi; Horvath, Steve

    2016-01-01

    Estimates of biological age based on DNA methylation patterns, often referred to as “epigenetic age”, “DNAm age”, have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2×10−9), independent of chronological age, even after adjusting for additional risk factors (p<5.4×10−4), and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5×10−43). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality. PMID:27690265

  15. Mitochondrial DNA mapping of social-biological interactions in Brazilian Amazonian African-descendant populations

    Directory of Open Access Journals (Sweden)

    Bruno Maia Carvalho

    2008-01-01

    Full Text Available The formation of the Brazilian Amazonian population has historically involved three main ethnic groups, Amerindian, African and European. This has resulted in genetic investigations having been carried out using classical polymorphisms and molecular markers. To better understand the genetic variability and the micro-evolutionary processes acting in human groups in the Brazilian Amazon region we used mitochondrial DNA to investigate 159 maternally unrelated individuals from five Amazonian African-descendant communities. The mitochondrial lineage distribution indicated a contribution of 50.2% from Africans (L0, L1, L2, and L3, 46.6% from Amerindians (haplogroups A, B, C and D and a small European contribution of 1.3%. These results indicated high genetic diversity in the Amerindian and African lineage groups, suggesting that the Brazilian Amazonian African-descendant populations reflect a possible population amalgamation of Amerindian women from different Amazonian indigenous tribes and African women from different geographic regions of Africa who had been brought to Brazil as slaves. The present study partially mapped the historical biological and social interactions that had occurred during the formation and expansion of Amazonian African-descendant communities.

  16. DNA methylation fingerprint of neuroblastoma reveals new biological and clinical insights

    Science.gov (United States)

    Gómez, Soledad; Castellano, Giancarlo; Mayol, Gemma; Queiros, Ana; Martín-Subero, José I.; Lavarino, Cinzia

    2015-01-01

    Neuroblastoma (NB) is one of the most frequently occurring extracranial solid tumors of childhood (Maris et al., 2007 [1]; Brodeur, 2003 [2]). Probability of cure varies according to patient's age, extent of disease and tumor biology (Maris et al., 2007 [1]; Brodeur, 2003 [2]; Cohn et al., 2009 [3]). However, the etiology of this developmental tumor is unknown. Recent evidence has shown that pediatric solid tumors, including NB, harbor a paucity of recurrent genetic mutations, with a significant proportion of recurrent events converging on epigenetic mechanisms (Cheung et al., 2012 [4]; Molenaar et al., 2012 [5]; Pugh et al., 2013 [6]; Sausen et al., 2013 [7]. We have analyzed the DNA methylome of neuroblastoma using high-density microarrays (Infinium Human Methylation 450k BeadChip) to define the epigenetic landscape of this pediatric tumor and its potential clinicopathological impact. Here, we provide the detail of methods and quality control parameters of the microarray data used for the study. Methylation data has been deposited at NCBI Gene Expression Omnibus data repository, accession number GSE54719; superseries record GSE54721. PMID:26484286

  17. Tracing the biological origin of animal glues used in paintings through mitochondrial DNA analysis.

    Science.gov (United States)

    Albertini, Emidio; Raggi, Lorenzo; Vagnini, Manuela; Sassolini, Alessandro; Achilli, Alessandro; Marconi, Gianpiero; Cartechini, Laura; Veronesi, Fabio; Falcinelli, Mario; Brunetti, Brunetto Giovanni; Miliani, Costanza

    2011-03-01

    We report the development of a suitable protocol for the identification of the biological origin of binding media on tiny samples from ancient paintings, by exploitation of the high specificity and high sensitivity offered by the state-of-the art DNA analysis. In particular, our aim was to molecularly characterize mitochondrial regions of the animal species traditionally employed for obtaining glues. The model has been developed using aged painting models and then tested to analyze the organic components in samples from the polychrome terracotta Madonna of Citerna by Donatello (1415-1420), where, by GC-MS and FTIR spectroscopy, animal glues and siccative oils were identified. The results obtained are good in terms of both sensibility and specificity of the method. First of all, it was possible to confirm that Donatello used animal glue for the preparation of the painted layers of the Madonna of Citerna and, specifically, glue derived from Bos taurus. Data obtained from sequencing confirm that each sample contains animal glue, revealing that it was mostly prepared from two common European taurine lineages called T2 and T3. There is one remarkable exception represented by one sample which falls into a surviving lineage of the now extinct European aurochs.

  18. Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer.

    Science.gov (United States)

    Murray, Sarah; McKenzie, Marian; Butler, Ruth; Baldwin, Samantha; Sutton, Kevin; Batey, Ian; Timmerman-Vaughan, Gail M

    2011-06-15

    Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.

  19. RNA2DNAlign: nucleotide resolution allele asymmetries through quantitative assessment of RNA and DNA paired sequencing data.

    Science.gov (United States)

    Movassagh, Mercedeh; Alomran, Nawaf; Mudvari, Prakriti; Dede, Merve; Dede, Cem; Kowsari, Kamran; Restrepo, Paula; Cauley, Edmund; Bahl, Sonali; Li, Muzi; Waterhouse, Wesley; Tsaneva-Atanasova, Krasimira; Edwards, Nathan; Horvath, Anelia

    2016-12-15

    We introduce RNA2DNAlign, a computational framework for quantitative assessment of allele counts across paired RNA and DNA sequencing datasets. RNA2DNAlign is based on quantitation of the relative abundance of variant and reference read counts, followed by binomial tests for genotype and allelic status at SNV positions between compatible sequences. RNA2DNAlign detects positions with differential allele distribution, suggesting asymmetries due to regulatory/structural events. Based on the type of asymmetry, RNA2DNAlign outlines positions likely to be implicated in RNA editing, allele-specific expression or loss, somatic mutagenesis or loss-of-heterozygosity (the first three also in a tumor-specific setting). We applied RNA2DNAlign on 360 matching normal and tumor exomes and transcriptomes from 90 breast cancer patients from TCGA. Under high-confidence settings, RNA2DNAlign identified 2038 distinct SNV sites associated with one of the aforementioned asymetries, the majority of which have not been linked to functionality before. The performance assessment shows very high specificity and sensitivity, due to the corroboration of signals across multiple matching datasets. RNA2DNAlign is freely available from http://github.com/HorvathLab/NGS as a self-contained binary package for 64-bit Linux systems.

  20. Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: comparison of two methods for DNA isolation.

    Science.gov (United States)

    Altamirano, Natalia Andrea; Rocco, Carlos; Aulicino, Paula; Sen, Luisa; Mangano, Andrea

    2010-12-01

    A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.

  1. Synthesis, biological investigation, calf thymus DNA binding and docking studies of the sulfonyl hydrazides and their derivatives

    Science.gov (United States)

    Murtaza, Shahzad; Shamim, Saima; Kousar, Naghmana; Tahir, Muhammad Nawaz; Sirajuddin, Muhammad; Rana, Usman Ali

    2016-03-01

    The present study describes the syntheses and biological investigations of sulfonyl hydrazides and their novel derivatives. The detailed investigations involved the characterization of the newly synthesized compounds using FTIR, NMR, mass spectrometry and by single crystal X-Ray diffraction (XRD) analysis techniques. The binding tendencies of these compounds with CT-DNA (calf thymus DNA) have been explored by electronic absorption (UV) spectroscopy and viscosity measurement. The binding constant (K) and Gibb's free energy (ΔG) values were also calculated accordingly. In addition, we also investigated the biological activities such as antioxidant, antibacterial, enzyme inhibition and DNA interactions. The antioxidant activity was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, while antibacterial activity was investigated against four bacterial strains (viz. Escherichia coli, Crynibacteria bovius, Staphylococcus auras and Bacillus antherasis) by employing the common disc diffusion method. Enzyme inhibition activity of the synthesized compounds was examined against butyrylcholinestrase. The results of enzyme inhibition activity and the DNA binding interaction studies were also collected through molecular docking program using computational analysis. Our study reveals that the newly synthesized compounds possess moderate to good biological activities.

  2. 3-Dimensional quantitative detection of nanoparticle content in biological tissue samples after local cancer treatment

    Energy Technology Data Exchange (ETDEWEB)

    Rahn, Helene, E-mail: helene.rahn@gmail.com [Institute of Fluid Mechanics, Chair of Magnetofluiddynamics, Technische Universitaet Dresden, Dresden 01069 (Germany); Alexiou, Christoph [ENT-Department, Section for Experimental Oncology and Nanomedicine (Else Kröner-Fresenius-Stiftungsprofessur), University Hospital Erlangen, Waldstraße 1, Erlangen 91054 (Germany); Trahms, Lutz [Physikalisch-Technische Bundesanstalt, Abbestraße 2-12, Berlin 10587 (Germany); Odenbach, Stefan [Institute of Fluid Mechanics, Chair of Magnetofluiddynamics, Technische Universitaet Dresden, Dresden 01069 (Germany)

    2014-06-01

    X-ray computed tomography is nowadays used for a wide range of applications in medicine, science and technology. X-ray microcomputed tomography (XµCT) follows the same principles used for conventional medical CT scanners, but improves the spatial resolution to a few micrometers. We present an example of an application of X-ray microtomography, a study of 3-dimensional biodistribution, as along with the quantification of nanoparticle content in tumoral tissue after minimally invasive cancer therapy. One of these minimal invasive cancer treatments is magnetic drug targeting, where the magnetic nanoparticles are used as controllable drug carriers. The quantification is based on a calibration of the XµCT-equipment. The developed calibration procedure of the X-ray-µCT-equipment is based on a phantom system which allows the discrimination between the various gray values of the data set. These phantoms consist of a biological tissue substitute and magnetic nanoparticles. The phantoms have been studied with XµCT and have been examined magnetically. The obtained gray values and nanoparticle concentration lead to a calibration curve. This curve can be applied to tomographic data sets. Accordingly, this calibration enables a voxel-wise assignment of gray values in the digital tomographic data set to nanoparticle content. Thus, the calibration procedure enables a 3-dimensional study of nanoparticle distribution as well as concentration. - Highlights: • Local cancer treatments are promising in reducing negative side effects occurring during conventional chemotherapy. • The nanoparticles play an important role in delivering drugs to the designated area during local cancer treatments as magnetic drug targeting. • We study the nanoparticles distribution in tumor tissue after magnetic drug targeting with X-ray computed tomography. • We achieved a 3-dimensional quantification of the nanoparticles content in tumor tissue out of digital tomographic data.

  3. Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

    Science.gov (United States)

    Chappell, James; Jensen, Kirsten; Freemont, Paul S.

    2013-01-01

    A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

  4. Role of quantitative and qualitative characteristics of free circulating DNA in the management of patients with non-small cell lung cancer.

    Science.gov (United States)

    Ulivi, Paola; Silvestrini, Rosella

    2013-12-01

    The release of DNA into peripheral blood is a common event in cancer patients, occurring as a consequence of necrotic and apoptotic processes typical of tumor cells. However, free circulating DNA (fcDNA) is also present in patients with benign diseases and in healthy individuals. Both quantitative and qualitative aspects of fcDNA have been studied as potential biomarkers in a number of tumor types. In particular, quantitative analysis of fcDNA has been shown to play an important role in the diagnosis of non-small cell lung cancer (NSCLC), because of its ability to discriminate between healthy subjects and individuals with NSCLC. Additionally, fcDNA in cancer patients derives predominantly from tumor tissue and, as such, it can be used for the molecular characterization of the primary tumor. Targeted therapies in NSCLC have, in recent years, produced promising results, highlighting the importance of molecular profiling of the primary cancer lesions. Considering that little or no tumor material is available for at least some of the patients, the possibility of using fcDNA for molecular analysis becomes increasingly important. In the present review we evaluated several quantitative and qualitative aspects of fcDNA that could be instrumental for the differential diagnosis of lung disease. There is ample evidence in the literature to support the possible use of peripheral blood-derived fcDNA in the early diagnosis and molecular characterization of lung cancer. This non-invasive method may also turn out to be valuable in monitoring drug response and in identifying induced mechanisms of drug resistance. Before it can be implemented in routine clinical practice, however, additional efforts are needed to standardize the methodology.

  5. A Parallel Biological Optimization Algorithm to Solve the Unbalanced Assignment Problem Based on DNA Molecular Computing.

    Science.gov (United States)

    Wang, Zhaocai; Pu, Jun; Cao, Liling; Tan, Jian

    2015-10-23

    The unbalanced assignment problem (UAP) is to optimally resolve the problem of assigning n jobs to m individuals (m parallel DNA algorithm for solving the unbalanced assignment problem using DNA molecular operations. We reasonably design flexible-length DNA strands representing different jobs and individuals, take appropriate steps, and get the solutions of the UAP in the proper length range and O(mn) time. We extend the application of DNA molecular operations and simultaneity to simplify the complexity of the computation.

  6. Detection and quantitation of benzo(a)pyrene-DNA adducts in brain and liver tissues of Beluga whales (Delphinapterus leucas) from the St. Lawrence and Mackenzie Estuaries

    Energy Technology Data Exchange (ETDEWEB)

    Shugart, L.R.

    1988-01-01

    It should be noted that there are few analytical techniques available for the detection and quantitation of chemical adducts in the DNA of living organisms. The reasons for this are: the analytical technique often has to accommodate the unique chemical and/or physical properties of the individual chemical or its metabolite; the percentage of total chemical that becomes most of the parent compound is usually detoxified and excreted; not all adducts that form between the genotoxic agent and DNA are stable or are involved in the development of subsequent deleterious events in the organism; and the amount of DNA available for analysis is often quite limited. 16 refs., 1 tab.

  7. DNA structure, binding mechanism and biology functions of polypyridyl complexes in biomedicine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    There is considerable research interest and vigorous debate about the DNA binding of polypyridyl complexes including the electron transfer involving DNA. In this review, based on the fluorescence quenching experiments, it was proposed that DNA might serve as a conductor. From the time-interval CD spectra, the different binding rates of D- and L-enantiomer to calf thymus DNA were observed. The factors influencing the DNA-binding of polypyridyl complexes, and the potential bio-functions of the complexes are also discussed.

  8. A fluorescent quantitative PCR method to detect Aspergillus fumigatus DNA%荧光定量PCR法检测烟曲霉DNA

    Institute of Scientific and Technical Information of China (English)

    毛璞; 余志辉; 黄红川; 杨淳; 黎毅敏

    2011-01-01

    Objective To develop a novel fluorescent quantitative PCR (qPCR) method to detect Aspergillus fumigatus (A. fumigatus) DNA, and evaluate its significance in diagnosing mouse invasive pulmonary aspergillosis. Methods Specific primers and fluorescent labeling probes were designed according to the sequence of internal transcribed spacer (ITS) regions of A. fumigatus. The qPCR was optimized and its specificity, amplification efficiency, sensitivity and repeatability were evaluated. The mouse model of A. fumigatusinfecting lung was established by using 36 adult male BALB/c mice with intratracheal injecting 1 × 106 ofA. fumigatus conidia. A. fumigatus DNA in the pulmonary tissue and bronchoalveolar lavage fluid (BALF) of the mice was measured by qPCR. Results The designed primers and probes were available for amplifying A. fumigatus DNA. The qPCR exhibited high sensitivity ( 100 conidia/mL) and wide range of linearity from 103 to 107 copies (R2 =0. 999). In concentrations of 0. 5 × 103 copies/mL and 0. 5 × 105 copies/mL, intra-assay coefficient of variation (CV) were 0. 39% and 0. 53%, while inter-assay CV were 2.22% and 4.58%, respectively. Conclusion A novel fluorescent qPCR method with high specificity, sensitivity and reproducibility was successfully established to detect A. fumigatus DNA. The method will be helpful for early diagnosis of A. fumigatus infection and observation of therapeutic efficacy.%目的 建立检测烟曲霉DNA的荧光定量PCR方法,评价其在小鼠侵袭性烟曲霉肺部感染诊断中的意义.方法 根据烟曲霉ITSI区域基因组序列,设计合成引物和荧光标记探针,优化荧光定量PCR条件,从特异性、扩增效率、灵敏度及重复性方面进行评价.建立小鼠烟曲霉肺部感染模型,用荧光定量PCR对36只实验小鼠肺组织及支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)进行检测.结果 本研究设计的引物和探针特异性扩增烟曲霉DNA

  9. A sensitive method to extract DNA from biological traces present on ammunition for the purpose of genetic profiling.

    Science.gov (United States)

    Dieltjes, Patrick; Mieremet, René; Zuniga, Sofia; Kraaijenbrink, Thirsa; Pijpe, Jeroen; de Knijff, Peter

    2011-07-01

    Exploring technological limits is a common practice in forensic DNA research. Reliable genetic profiling based on only a few cells isolated from trace material retrieved from a crime scene is nowadays more and more the rule rather than the exception. On many crime scenes, cartridges, bullets, and casings (jointly abbreviated as CBCs) are regularly found, and even after firing, these potentially carry trace amounts of biological material. Since 2003, the Forensic Laboratory for DNA Research is routinely involved in the forensic investigation of CBCs in the Netherlands. Reliable DNA profiles were frequently obtained from CBCs and used to match suspects, victims, or other crime scene-related DNA traces. In this paper, we describe the sensitive method developed by us to extract DNA from CBCs. Using PCR-based genotyping of autosomal short tandem repeats, we were able to obtain reliable and reproducible DNA profiles in 163 out of 616 criminal cases (26.5%) and in 283 out of 4,085 individual CBC items (6.9%) during the period January 2003-December 2009. We discuss practical aspects of the method and the sometimes unexpected effects of using cell lysis buffer on the subsequent investigation of striation patterns on CBCs.

  10. Assembly of Designed Oligonucleotides: a useful tool in synthetic biology for creating high-quality combinatorial DNA libraries.

    Science.gov (United States)

    Acevedo-Rocha, Carlos G; Reetz, Manfred T

    2014-01-01

    The method dubbed Assembly of Designed Oligonucleotides (ADO) is a powerful tool in synthetic biology to create combinatorial DNA libraries for gene, protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The first involves a primer-free, polymerase cycling assembly or overlap extension PCR step using carefully designed overlapping oligonucleotides. The second step is a PCR amplification using the outer primers, resulting in a high-quality and bias-free double-stranded DNA library that can be assembled with other gene fragments and/or cloned into a suitable plasmid subsequently. The protocol can be performed in a few hours. In theory, neither the length of the DNA library nor the number of DNA changes has any limits. Furthermore, with the costs of synthetic DNA dropping every year, after an initial investment is made in the oligonucleotides, these can be exchanged for alternative ones with different sequences at any point in the process, fully exploiting the potential of creating highly diverse combinatorial libraries. In the example chosen here, we show the construction of a high-quality combinatorial ADO library targeting sixteen different codons simultaneously with nonredundant degenerate codons encoding various reduced alphabets of four amino acids along the heme region of the monooxygenase P450-BM3.

  11. Quantitation of cytomegalovirus (CMV) DNA in leukocytes of human immunodeficiency virus-infected subjects with and without CMV disease by using PCR and the SHARP Signal Detection System.

    Science.gov (United States)

    Boivin, G; Handfield, J; Murray, G; Toma, E; Lalonde, R; Lazar, J G; Bergeron, M G

    1997-02-01

    We report the development of a simple and rapid PCR assay for quantitation of the cytomegalovirus (CMV) DNA load in polymorphonuclear leukocytes. Using this system, a very good correlation was found between a high number of CMV copies in the blood and the presence of CMV disease in subjects with AIDS.

  12. Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

    Science.gov (United States)

    Liu, Shuo; Ji, Debin; Cliffe, Laura; Sabatini, Robert; Wang, Yinsheng

    2014-10-01

    β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the

  13. Structural Biology of DNA Repair: Spatial Organisation of the Multicomponent Complexes of Nonhomologous End Joining

    Directory of Open Access Journals (Sweden)

    Takashi Ochi

    2010-01-01

    Full Text Available Nonhomologous end joining (NHEJ plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods—X-ray crystallography, electron microscopy and small angle X-ray scattering—can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere.

  14. Palladium(II) complexes as biologically potent metallo-drugs: Synthesis, spectral characterization, DNA interaction studies and antibacterial activity

    Science.gov (United States)

    Prasad, Kollur Shiva; Kumar, Linganna Shiva; Chandan, Shivamallu; Naveen Kumar, R. M.; Revanasiddappa, Hosakere D.

    2013-04-01

    Four novel mononuclear Pd(II) complexes have been synthesized with the biologically active Schiff base ligands (L1-L4) derived from 3-amino-2-methyl-4(3H)-quinazolinone. The structure of the complexes has been proposed by elemental analysis, molar conductance, IR, 1H NMR, mass, UV-Vis spectrometric and thermal studies. The investigation of interaction of the complexes with calf thymus DNA (CT-DNA) has been performed with absorption and fluorescence spectroscopic studies. The nuclease activity was done using pUC19 supercoiled DNA by gel-electrophoresis. All the ligands and their Pd(II) complexes have also been screened for their antibacterial activity by discolor diffusion technique.

  15. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part A: Eukaryotic Gene Structure and DNA Replication

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available Progress in the basic sciences of cell and molecular biology has provided an exciting dimension that has translated into clinically relevant information in every medical subspecialty. Importantly, the application of recombinant DNA technology has played a major role in unravelling the intricacies related to the molecular pathophysiology of disease. This series of review articles constitutes a framework for the integration of the database of new information into the core knowledge base of concepts related to the pathogenesis of gastrointestinal disorders and liver disease. The goal of this series of three articles is to review the basic principles of eukaryotic gene expression. The first article examines the role of DNA in directing the flow of genetic information in eukaryotic cells.

  16. Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX.

    Science.gov (United States)

    Savory, Nasa; Nzakizwanayo, Jonathan; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Dedi, Cinzia; Jones, Brian V; Ikebukuro, Kazunori

    2014-09-01

    In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli. Copyright © 2014. Published by Elsevier B.V.

  17. Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma.

    Science.gov (United States)

    Cao, Yuwen; Li, Yixiao; Zhang, Na; Hu, Jianming; Yin, Liang; Pan, Zemin; Li, Yucong; Du, Xiaoming; Zhang, Wenjie; Li, Feng

    2015-01-01

    Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer.

  18. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    Science.gov (United States)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure assessment.

  19. Quantitative assessment of DNA methylation for the detection of cervical and endometrial adenocarcinomas in liquid-based cytology specimens.

    Science.gov (United States)

    Kim, Ga-Eon; Kweon, Sun-Seog; Lee, Ji Shin; Lee, Jae Hyuk; Nam, Jong Hee; Choi, Chan

    2012-08-01

    To investigate the aberrant promoter hypermethylation as a screening tool for cervical adenocarcinomas (CAs) and endometrial adenocarcinomas (EAs) in cervical scrapings. A quantitative multiplex methylation-specific polymerase chain reaction approach was used to examine promoter methylation of 5 genes (APC, HIN-1, RAR-beta, RASSF1A and Twist) in biopsy-confirmed CA (n = 31) and EA (n = 27) residual, liquid-based cytology samples. The data of negative for intraepithelial lesions or malignancy and low-grade squamous intraepithelial lesions were used as controls. Methylation levels of APC, RAR-beta, RASSF1A and Twist were significantly higher in CA than in control cervical samples. For EA, only the methylation levels of RASSF1A differed significantly from those of control. Receiver-operating characteristic analysis demonstrated that APC, RAR-beta and RASSF1A had the ability to distinguish CA/EA, CA and EA from control samples. In CA/EA and CA samples, the best 3-gene combination was RASSF1A/RAR-beta/APC. This 3-gene panel had a sensitivity of 87.0% for CA/EA and of 80.6% for CA and a specificity of 79.3% for both CA/EA and CA. In EA samples, RASSF1A showed the best performance in distinguishing EA from control. The estimated sensitivity of RASSF1A for detecting EA was 63.0%, and its specificity was 96.3%. This feasibility study demonstrates that quantitative detection of aberrant DNA methylation in cervical scrapings may be a promising new diagnostic tool for the detection of CA and EA.

  20. Leo Szilard Lectureship Award Talk - Universal Scaling Laws from Cells to Cities; A Physicist's Search for Quantitative, Unified Theories of Biological and Social Structure and Dynamics

    Science.gov (United States)

    West, Geoffrey

    2013-04-01

    Many of the most challenging, exciting and profound questions facing science and society, from the origins of life to global sustainability, fall under the banner of ``complex adaptive systems.'' This talk explores how scaling can be used to begin to develop physics-inspired quantitative, predictive, coarse-grained theories for understanding their structure, dynamics and organization based on underlying mathematisable principles. Remarkably, most physiological, organisational and life history phenomena in biology and socio-economic systems scale in a simple and ``universal'' fashion: metabolic rate scales approximately as the 3/4-power of mass over 27 orders of magnitude from complex molecules to the largest organisms. Time-scales (such as lifespans and growth-rates) and sizes (such as genome lengths and RNA densities) scale with exponents which are typically simple multiples of 1/4, suggesting that fundamental constraints underlie much of the generic structure and dynamics of living systems. These scaling laws follow from dynamical and geometrical properties of space-filling, fractal-like, hierarchical branching networks, presumed optimised by natural selection. This leads to a general framework that potentially captures essential features of diverse systems including vasculature, ontogenetic growth, cancer, aging and mortality, sleep, cell size, and DNA nucleotide substitution rates. Cities and companies also scale: wages, profits, patents, crime, disease, pollution, road lengths scale similarly across the globe, reflecting underlying universal social network dynamics which point to general principles of organization transcending their individuality. These have dramatic implications for global sustainability: innovation and wealth creation that fuel social systems, left unchecked, potentially sow the seeds for their inevitable collapse.

  1. Internal-Modified Dithiol DNA-Directed Au Nanoassemblies: Geometrically Controlled Self-Assembly and Quantitative Surface-Enhanced Raman Scattering Properties

    Science.gov (United States)

    Yan, Yuan; Shan, Hangyong; Li, Min; Chen, Shu; Liu, Jianyu; Cheng, Yanfang; Ye, Cui; Yang, Zhilin; Lai, Xuandi; Hu, Jianqiang

    2015-11-01

    In this work, a hierarchical DNA-directed self-assembly strategy to construct structure-controlled Au nanoassemblies (NAs) has been demonstrated by conjugating Au nanoparticles (NPs) with internal-modified dithiol single-strand DNA (ssDNA) (Au-B-A or A-B-Au-B-A). It is found that the dithiol-ssDNA-modified Au NPs and molecule quantity of thiol-modified ssDNA grafted to Au NPs play critical roles in the assembly of geometrically controlled Au NAs. Through matching Au-DNA self-assembly units, geometrical structures of the Au NAs can be tailored from one-dimensional (1D) to quasi-2D and 2D. Au-B-A conjugates readily give 1D and quasi-2D Au NAs while 2D Au NAs can be formed by A-B-Au-B-A building blocks. Surface-enhanced Raman scattering (SERS) measurements and 3D finite-difference time domain (3D-FDTD) calculation results indicate that the geometrically controllable Au NAs have regular and linearly “hot spots”-number-depended SERS properties. For a certain number of NPs, the number of “hot spots” and accordingly enhancement factor of Au NAs can be quantitatively evaluated, which open a new avenue for quantitative analysis based on SERS technique.

  2. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  3. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

    Science.gov (United States)

    Malik, Afshan N; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  4. A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles.

    Science.gov (United States)

    Nakae, Yoshiko; Stoward, Peter J; Bespalov, Ivan A; Melamede, Robert J; Wallace, Susan S

    2005-09-01

    This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases

  5. QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACID LEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

    NARCIS (Netherlands)

    WITHOFF, S; SMIT, EF; MEERSMA, GJ; van den Berg, Anke; TIMMERBOSSCHA, H; KOK, K; POSTMUS, PE; MULDER, NH; DEVRIES, EGE; BUYS, CHCM

    1994-01-01

    BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase I

  6. The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response — Biological and Clinical Aspects

    Energy Technology Data Exchange (ETDEWEB)

    Medová, Michaela, E-mail: michaela.medova@dkf.unibe.ch; Aebersold, Daniel M.; Zimmer, Yitzhak, E-mail: michaela.medova@dkf.unibe.ch [Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, 3010 Bern (Switzerland); Department of Clinical Research, University of Bern, DKF, MEM-E807, Murtenstrasse 35, 3010 Bern (Switzerland)

    2013-12-19

    Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

  7. Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

    Science.gov (United States)

    Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

    2016-04-01

    Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

  8. Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

    Science.gov (United States)

    Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

    2010-01-01

    This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

  9. Evaluation of DNA dosimetry to assess ozone-mediated variability of biologically harmful radiation in Antarctica

    NARCIS (Netherlands)

    George, AL; Peat, HJ; Buma, AGJ

    2002-01-01

    In this study we investigated the use of a DNA dosimeter to accurately measure changes in ultraviolet B radiation (UVBR; 280-315 nm) under Antarctic ozone hole conditions. Naked DNA solution in quartz tubes was exposed to ambient solar radiation at Rothera Research Station, Antarctica, between Octob

  10. Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

    Science.gov (United States)

    Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

    2010-01-01

    This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

  11. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    Science.gov (United States)

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.

  12. Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yarosh, Daniel B

    2002-11-30

    The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

  13. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  14. Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

    Science.gov (United States)

    Kam, Winnie W. Y.; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. PMID:23722662

  15. DNA adductomics.

    Science.gov (United States)

    Balbo, Silvia; Turesky, Robert J; Villalta, Peter W

    2014-03-17

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the (32)P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC-MS(n)), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC-MS(n) instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.

  16. Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

    Science.gov (United States)

    Baluda, M A; Shoyab, M; Evans, R; Markham, P D; Ali, M

    1975-01-01

    Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.

  17. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

    Science.gov (United States)

    Acharya, Kamal R; Dhand, Navneet K; Whittington, Richard J; Plain, Karren M

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

  18. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

    Science.gov (United States)

    Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

  19. Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

    Science.gov (United States)

    Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

    2017-05-15

    Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection.

  20. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  1. From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments

    Science.gov (United States)

    Recent technological advances have driven rapid development of DNA-based methods designed to facilitate detection and monitoring of invasive species in aquatic environments. These tools promise to significantly alleviate difficulties associated with traditional monitoring approac...

  2. From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments

    Science.gov (United States)

    Recent technological advances have driven rapid development of DNA-based methods designed to facilitate detection and monitoring of invasive species in aquatic environments. These tools promise to significantly alleviate difficulties associated with traditional monitoring approac...

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

    Science.gov (United States)

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

    2013-05-23

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

  4. Biological effects of DNA damage in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reilly, Michelle S; Grogan, Dennis W

    2002-02-19

    To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.

  5. Collaborative Learning in Biology: Debating the Ethics of Recombinant DNA Technology.

    Science.gov (United States)

    Anderson, Rodney P.

    1998-01-01

    Discusses applications of recombinant DNA technology and the controversies surrounding that technique. Provides a cooperative learning project idea that involves teams of students investigating and debating these issues. (DDR)

  6. A Parallel Biological Optimization Algorithm to Solve the Unbalanced Assignment Problem Based on DNA Molecular Computing

    Directory of Open Access Journals (Sweden)

    Zhaocai Wang

    2015-10-01

    Full Text Available The unbalanced assignment problem (UAP is to optimally resolve the problem of assigning n jobs to m individuals (m < n, such that minimum cost or maximum profit obtained. It is a vitally important Non-deterministic Polynomial (NP complete problem in operation management and applied mathematics, having numerous real life applications. In this paper, we present a new parallel DNA algorithm for solving the unbalanced assignment problem using DNA molecular operations. We reasonably design flexible-length DNA strands representing different jobs and individuals, take appropriate steps, and get the solutions of the UAP in the proper length range and O(mn time. We extend the application of DNA molecular operations and simultaneity to simplify the complexity of the computation.

  7. DNAtraffic—a new database for systems biology of DNA dynamics during the cell life

    OpenAIRE

    Kuchta, Krzysztof; Barszcz, Daniela; Grzesiuk, Elzbieta; Pomorski, Pawel; Krwawicz, Joanna

    2011-01-01

    DNAtraffic (http://dnatraffic.ibb.waw.pl/) is dedicated to be a unique comprehensive and richly annotated database of genome dynamics during the cell life. It contains extensive data on the nomenclature, ontology, structure and function of proteins related to the DNA integrity mechanisms such as chromatin remodeling, histone modifications, DNA repair and damage response from eight organisms: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae,...

  8. Environmental DNA Marker Development with Sparse Biological Information: A Case Study on Opossum Shrimp (Mysis diluviana).

    Science.gov (United States)

    Carim, Kellie J; Christianson, Kyle R; McKelvey, Kevin M; Pate, William M; Silver, Douglas B; Johnson, Brett M; Galloway, Bill T; Young, Michael K; Schwartz, Michael K

    2016-01-01

    The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth.

  9. 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.

  10. A DNA methylation-based definition of biologically distinct breast cancer subtypes.

    Science.gov (United States)

    Stefansson, Olafur A; Moran, Sebastian; Gomez, Antonio; Sayols, Sergi; Arribas-Jorba, Carlos; Sandoval, Juan; Hilmarsdottir, Holmfridur; Olafsdottir, Elinborg; Tryggvadottir, Laufey; Jonasson, Jon G; Eyfjord, Jorunn; Esteller, Manel

    2015-03-01

    In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis.

  11. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, A. Viviana, E-mail: alicia.pinto@incqs.fiocruz.br [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Deodato, Elder L. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Cardoso, Janine S. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Leitao, Alvaro C. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Padula, Marcelo de [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil)

    2010-06-01

    Although titanium dioxide (TiO{sub 2}) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO{sub 2} is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO{sub 2}-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO{sub 2} associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO{sub 2} plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO{sub 2} protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO{sub 2} plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO{sub 2} plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  12. Potential bioactive Schiff base compounds: Synthesis, characterization, X-ray structures, biological screenings and interaction with Salmon sperm DNA

    Science.gov (United States)

    Sirajuddin, Muhammad; Uddin, Noor; Ali, Saqib; Tahir, Muhammad Nawaz

    2013-12-01

    Three Schiff base compounds ofN‧-substituted benzohydrazide and sulfonohydrazide derivatives: N‧-(2-hydroxy-3-methoxybenzylidene)-4-tert-butyl- benzohydrazide (1), N‧-(5-bromo-2-hydroxybenzylidene)-4-tert-butylbenzohydrazide (2) and N‧-(2-hydroxy-3-methoxybenzylidene)-4-methylbenzenesulfonohydrazide (3) were synthesized and characterized by elemental analysis, FT-IR, 1H, 13C NMR spectroscopy and single crystal analysis. The title compounds have been screened for their biological activities including, antibacterial, antifungal, antioxidant, cytotoxic, enzymatic activities as well as interaction with SS-DNA which showed remarkable activities in each area of research. The DNA binding of the compounds 1-3 with SS-DNA has been carried out with absorption spectroscopy, which reveals the binding propensity towards SS-DNA via intercalation mode of interaction. The intercalative mode of interaction is also supported by viscometric results. The synthesized compounds were also found to be effective against alkaline phosphatase enzyme. They also show significant to good antimicrobial activity against six bacterial and five fungal strains. The MIC (minimum inhibitory concentration) for antibacterial activity ranges from 1.95-500 μg/mL. Compounds 1-3 show cytotoxic activity comparable to the control. At higher conc. (100 μg/L) compound 3 shows 100% activity means that it has killed all brine shrimps. They were also found to be effective antioxidant of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and show almost comparable antioxidant activity to that of the standard and known antioxidant, ascorbic acid.

  13. Synthesis, structural characterization and biological activity of a trinuclear zinc(II) complex: DNA interaction study and antimicrobial activity

    Indian Academy of Sciences (India)

    Bhaskar Biswas; Niranjan Kole; Moumita Patra; Shampa Dutta; Mousumi Ganguly

    2013-11-01

    A trinuclear zinc(II) complex [Zn3L2(-O2CCH3)2(H2O)2]·H2O·2CH3OH (1) was synthesized from an in situ reaction between zinc acetate and a Schiff base ligand (H2L = 2-((2-hydroxyphenylimino) methyl)-6-methoxyphenol). The ligand was prepared by (1:1) condensation of ortho-vanillin and ortho-aminophenol. The ligand and zinc(II) complex were characterized by elemental analysis, Fourier Transform Infrared (FTIR), 1H-Nuclear Magnetic Resonance (NMR), UV-Vis spectroscopy, Powder X-ray Diffraction (PXRD) and thermogravimetric analysis. 1 crystallizes in P-1 space group with = 11.9241(3) Å, = 12.19746 Å, = 20.47784 Å with unit cell volume is 2674.440 (Å)3. Binding property of the complex with calf thymus DNA (CT-DNA) has been investigated using absorption and emission studies. Thermal melting and viscosity experiments were further performed to determine the mode of binding of 1 with CT-DNA. Spectroscopic and viscosity investigations revealed an intercalative binding mode of 1 with CT-DNA. The ligand and its zinc complex were screened for their biological activity against bacterial species and fungi. Activity data show that the metal complex has more antibacterial and antifungal activity than the parent Schiff base ligand and against those bacterial or fungi species.

  14. Z-DNA and its biological function%Z-DNA及其生物学功能

    Institute of Scientific and Technical Information of China (English)

    汤雅男; 杨攀; 胡成钰

    2009-01-01

    Z-DNA是一种处于高能状态、不稳定的DNA分子构象.形成Z-DNA的原因有很多:首先,转录过程中,移动的RNA聚合酶在模板DNA的5'端产生负超螺旋扭曲力,导致Z-DNA的形成;其次,含有d(GC)_n序列的核酸分子在高浓度的NaCl、[Co(NH_3)_6]~(2+)盐溶液