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.

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Science.gov (United States)

Wu, Min; Li, Jin; Yue, Lei; Bai, Lu; Li, Yaming; Chen, Jieliang; Zhang, Xiaonan; Yuan, Zhenghong

2018-04-01

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Development and validation of open-source software for DNA mixture interpretation based on a quantitative continuous model.

Science.gov (United States)

Manabe, Sho; Morimoto, Chie; Hamano, Yuya; Fujimoto, Shuntaro; Tamaki, Keiji

2017-01-01

In criminal investigations, forensic scientists need to evaluate DNA mixtures. The estimation of the number of contributors and evaluation of the contribution of a person of interest (POI) from these samples are challenging. In this study, we developed a new open-source software "Kongoh" for interpreting DNA mixture based on a quantitative continuous model. The model uses quantitative information of peak heights in the DNA profile and considers the effect of artifacts and allelic drop-out. By using this software, the likelihoods of 1-4 persons' contributions are calculated, and the most optimal number of contributors is automatically determined; this differs from other open-source software. Therefore, we can eliminate the need to manually determine the number of contributors before the analysis. Kongoh also considers allele- or locus-specific effects of biological parameters based on the experimental data. We then validated Kongoh by calculating the likelihood ratio (LR) of a POI's contribution in true contributors and non-contributors by using 2-4 person mixtures analyzed through a 15 short tandem repeat typing system. Most LR values obtained from Kongoh during true-contributor testing strongly supported the POI's contribution even for small amounts or degraded DNA samples. Kongoh correctly rejected a false hypothesis in the non-contributor testing, generated reproducible LR values, and demonstrated higher accuracy of the estimated number of contributors than another software based on the quantitative continuous model. Therefore, Kongoh is useful in accurately interpreting DNA evidence like mixtures and small amounts or degraded DNA samples.

Development and validation of open-source software for DNA mixture interpretation based on a quantitative continuous model.

Directory of Open Access Journals (Sweden)

Sho Manabe

Full Text Available In criminal investigations, forensic scientists need to evaluate DNA mixtures. The estimation of the number of contributors and evaluation of the contribution of a person of interest (POI from these samples are challenging. In this study, we developed a new open-source software "Kongoh" for interpreting DNA mixture based on a quantitative continuous model. The model uses quantitative information of peak heights in the DNA profile and considers the effect of artifacts and allelic drop-out. By using this software, the likelihoods of 1-4 persons' contributions are calculated, and the most optimal number of contributors is automatically determined; this differs from other open-source software. Therefore, we can eliminate the need to manually determine the number of contributors before the analysis. Kongoh also considers allele- or locus-specific effects of biological parameters based on the experimental data. We then validated Kongoh by calculating the likelihood ratio (LR of a POI's contribution in true contributors and non-contributors by using 2-4 person mixtures analyzed through a 15 short tandem repeat typing system. Most LR values obtained from Kongoh during true-contributor testing strongly supported the POI's contribution even for small amounts or degraded DNA samples. Kongoh correctly rejected a false hypothesis in the non-contributor testing, generated reproducible LR values, and demonstrated higher accuracy of the estimated number of contributors than another software based on the quantitative continuous model. Therefore, Kongoh is useful in accurately interpreting DNA evidence like mixtures and small amounts or degraded DNA samples.

Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

DEFF Research Database (Denmark)

Zhan, Zongrui; Cao, Cuong; Sim, Sang Jun

2010-01-01

Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of go...... to the concentration of the target DNA, could easily be confirmed by a UV–vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method....

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…

Infusion of Quantitative and Statistical Concepts into Biology Courses Does Not Improve Quantitative Literacy

Science.gov (United States)

Beck, Christopher W.

2018-01-01

Multiple national reports have pushed for the integration of quantitative concepts into the context of disciplinary science courses. The aim of this study was to evaluate the quantitative and statistical literacy of biology students and explore learning gains when those skills were taught implicitly in the context of biology. I examined gains in…

A rapid and quantitative method to determine the tritium content in DNA from small tissue sampes

International Nuclear Information System (INIS)

Kasche, V.; Zoellner, R.

1979-01-01

A rapid and quantitative two-step procedure to isolate double-strand DNA from small (10-100 mg) animal tissue samples is presented. The method is developed for investigations to evaluate the relative importance of organically bound tritium for the dose factors used to calculate dose commitments due to this nuclide. In the first step the proteins in the homogenized sample are hydrolysed, at a high pH (9.0) and ionic strength (1.5) to dissociate protein from DNA, using immobilized Proteinase K as a proteolytic enzyme. The DNA is then absorbed to hydroxylapatite and separated from impurities by step-wise elution with buffers of increasing ionic strength. More than 90% of the DNA in the samples could be isolated in double-strand form by this procedure. The method has been applied to determine pool-sizes and biological half-life times of tritium in DNA from various animal (mouse) tissues. It has also been shown to be suitable in other radiobiological studies where effects on DNA are investigated. (author)

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.

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. PMID:24591504

Quantitative analysis of gene-specific DNA damage in human spermatozoa

International Nuclear Information System (INIS)

Sawyer, Dennis E.; Mercer, Belinda G.; Wiklendt, Agnieszka M.; Aitken, R. John

2003-01-01

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H 2 O 2 ; 0-5 mM) or iron (as Fe(II)SO 4 , 0-500 μM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, β-pol and β-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H 2 O 2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H 2 O 2 . The mitochondrial genome of human spermatozoa was significantly (P 2 O 2 -induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage

Developing a biological dosimeter based on mitochondrial DNA

Energy Technology Data Exchange (ETDEWEB)

Adams, S; Carlisle, S M; Unrau, P; Deugau, K V [Atomic Energy of Canada Ltd., Chalk River, ON (Canada)

1996-12-31

Direct measurement of deoxyribonucleic acid (DNA) damage from ionizing radiation may be advantageous in determining radiation radiation exposures and assessing their effects on atomic radiation workers. The mitochondrial DNA molecule is one potential cellular DNA target which is: fully defined and sequenced; present in many copies per cell; not vital to cellular survival; and less subject to DNA repair than nuclear DNA. A method is described to isolate and analyse normal mitochondrial DNA. We describe the developments needed to determine DNA damage in mitochondrial DNA. The target is to make a biological dosimeter. (author). 6 refs., 3 figs.

Developing a biological dosimeter based on mitochondrial DNA

International Nuclear Information System (INIS)

Adams, S.; Carlisle, S.M.; Unrau, P.; Deugau, K.V.

1995-01-01

Direct measurement of deoxyribonucleic acid (DNA) damage from ionizing radiation may be advantageous in determining radiation radiation exposures and assessing their effects on atomic radiation workers. The mitochondrial DNA molecule is one potential cellular DNA target which is: fully defined and sequenced; present in many copies per cell; not vital to cellular survival; and less subject to DNA repair than nuclear DNA. A method is described to isolate and analyse normal mitochondrial DNA. We describe the developments needed to determine DNA damage in mitochondrial DNA. The target is to make a biological dosimeter. (author). 6 refs., 3 figs

High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

CSIR Research Space (South Africa)

Van den Berg, N

2004-11-01

Full Text Available Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression...

Fluorescence quantitative PCR in detection of HBV DNA

International Nuclear Information System (INIS)

Shen Zheng; Li Ming; Shen Xia

2003-01-01

Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

Development of a Biological Science Quantitative Reasoning Exam (BioSQuaRE)

Science.gov (United States)

Stanhope, Liz; Ziegler, Laura; Haque, Tabassum; Le, Laura; Vinces, Marcelo; Davis, Gregory K.; Zieffler, Andrew; Brodfuehrer, Peter; Preest, Marion; Belitsky, Jason M.; Umbanhowar, Charles, Jr.; Overvoorde, Paul J.

2017-01-01

Multiple reports highlight the increasingly quantitative nature of biological research and the need to innovate means to ensure that students acquire quantitative skills. We present a tool to support such innovation. The Biological Science Quantitative Reasoning Exam (BioSQuaRE) is an assessment instrument designed to measure the quantitative…

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... 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...

Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

Energy Technology Data Exchange (ETDEWEB)

Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

1994-09-01

The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

Lesion measurement in non-radioactive DNA by quantitative gel electrophoresis

International Nuclear Information System (INIS)

Sutherland, J.C.; Chen, Chun Zhang; Emrick, A.; Hacham, H; Monteleone, D.; Ribeiro, E.; Trunk, J.; Sutherland, B.M.

1989-01-01

The gel electrophoresis method developed during the past ten years in our laboratories makes possible the quantitation of UV induced pyrimidine dimers, gamma ray induced single- and double-strand breaks and many other types of lesions in nanogram quantities of DNA. The DNA does not have to be labeled with radionuclides or of a particular conformation, thus facilitating the use of the method in measuring damage levels and repair rates in the DNA of intact organisms -- including man. The gel method can quantitate any lesion in DNA that either is, or can be converted to a single- or double-strand break. The formation of a strand break produces two shorter DNA molecules for each molecule that existed before the treatment that produced the break. Determining the number of breaks, and hence the number of lesions, becomes a matter of comparing the average lengths of molecules in samples differing only in lesion-induced breaks. This requires that we determine the distribution of mass of DNA on a gel as a function of its distance of migration and also the dispersion function of its distance of migration and also the dispersion function (the relationship between molecular length and distance of migration) in the gel electrophoresis system. 40 refs., 5 figs

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

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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Extraction of DNA from Forensic Biological Samples for Genotyping.

Science.gov (United States)

Stray, J E; Liu, J Y; Brevnov, M G; Shewale, J G

2010-07-01

Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms. Copyright © 2010 Central Police University.

Population variability in biological adaptive responses to DNA damage and the shapes of carcinogen dose-response curves

International Nuclear Information System (INIS)

Conolly, Rory B.; Gaylor, David W.; Lutz, Werner K.

2005-01-01

Carcinogen dose-response curves for both ionizing radiation and chemicals are typically assumed to be linear at environmentally relevant doses. This assumption is used to ensure protection of the public health in the absence of relevant dose-response data. A theoretical justification for the assumption has been provided by the argument that low dose linearity is expected when an exogenous agent adds to an ongoing endogenous process. Here, we use computational modeling to evaluate (1) how two biological adaptive processes, induction of DNA repair and cell cycle checkpoint control, may affect the shapes of dose-response curves for DNA-damaging carcinogens and (2) how the resulting dose-response behaviors may vary within a population. Each model incorporating an adaptive process was capable of generating not only monotonic dose-responses but also nonmonotonic (J-shaped) and threshold responses. Monte Carlo analysis suggested that all these dose-response behaviors could coexist within a population, as the spectrum of qualitative differences arose from quantitative changes in parameter values. While this analysis is largely theoretical, it suggests that (a) accurate prediction of the qualitative form of the dose-response requires a quantitative understanding of the mechanism (b) significant uncertainty is associated with human health risk prediction in the absence of such quantitative understanding and (c) a stronger experimental and regulatory focus on biological mechanisms and interindividual variability would allow flexibility in regulatory treatment of environmental carcinogens without compromising human health

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.

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

International Nuclear Information System (INIS)

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

Advances in forensic DNA quantification: a review.

Science.gov (United States)

Lee, Steven B; McCord, Bruce; Buel, Eric

2014-11-01

This review focuses upon a critical step in forensic biology: detection and quantification of human DNA from biological samples. Determination of the quantity and quality of human DNA extracted from biological evidence is important for several reasons. Firstly, depending on the source and extraction method, the quality (purity and length), and quantity of the resultant DNA extract can vary greatly. This affects the downstream method as the quantity of input DNA and its relative length can determine which genotyping procedure to use-standard short-tandem repeat (STR) typing, mini-STR typing or mitochondrial DNA sequencing. Secondly, because it is important in forensic analysis to preserve as much of the evidence as possible for retesting, it is important to determine the total DNA amount available prior to utilizing any destructive analytical method. Lastly, results from initial quantitative and qualitative evaluations permit a more informed interpretation of downstream analytical results. Newer quantitative techniques involving real-time PCR can reveal the presence of degraded DNA and PCR inhibitors, that provide potential reasons for poor genotyping results and may indicate methods to use for downstream typing success. In general, the more information available, the easier it is to interpret and process the sample resulting in a higher likelihood of successful DNA typing. The history of the development of quantitative methods has involved two main goals-improving precision of the analysis and increasing the information content of the result. This review covers advances in forensic DNA quantification methods and recent developments in RNA quantification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

Science.gov (United States)

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

Chemical determination of free radical-induced damage to DNA.

Science.gov (United States)

Dizdaroglu, M

1991-01-01

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

Science.gov (United States)

Dash, Hirak Ranjan; Das, Surajit

2018-02-01

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

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.

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.

Interfacing DNA nanodevices with biology: challenges, solutions and perspectives

International Nuclear Information System (INIS)

Vinther, Mathias; Kjems, Jørgen

2016-01-01

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

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

Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

International Nuclear Information System (INIS)

Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

1989-01-01

A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

Science.gov (United States)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

International Nuclear Information System (INIS)

Cao Hongbin; Banh, Alice; Kwok, Shirley; Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha; Bala, Rajeev; Pinsky, Benjamin A.; Colevas, Dimitrios; Pourmand, Nader; Koong, Albert C.; Kong, Christina S.; Le, Quynh-Thu

2012-01-01

Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16 INK4a staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using 18 F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

Quantitative analysis of the flexibility effect of cisplatin on circular DNA

Science.gov (United States)

Ji, Chao; Zhang, Lingyun; Wang, Peng-Ye

2013-10-01

We study the effects of cisplatin on the circular configuration of DNA using atomic force microscopy (AFM) and observe that the DNA gradually transforms to a complex configuration with an intersection and interwound structures from a circlelike structure. An algorithm is developed to extract the configuration profiles of circular DNA from AFM images and the radius of gyration is used to describe the flexibility of circular DNA. The quantitative analysis of the circular DNA demonstrates that the radius of gyration gradually decreases and two processes on the change of flexibility of circular DNA are found as the cisplatin concentration increases. Furthermore, a model is proposed and discussed to explain the mechanism for understanding the complicated interaction between DNA and cisplatin.

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.

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.

2015-01-01

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. PMID:26067805

DNA agarose gel electrophoresis for antioxidant analysis: Development of a quantitative approach for phenolic extracts.

Science.gov (United States)

Silva, Sara; Costa, Eduardo M; Vicente, Sandra; Veiga, Mariana; Calhau, Conceição; Morais, Rui M; Pintado, Manuela E

2017-10-15

Most of the fast in vitro assays proposed to determine the antioxidant capacity of a compound/extract lack either biological context or employ complex protocols. Therefore, the present work proposes the improvement of an agarose gel DNA electrophoresis in order to allow for a quantitative estimation of the antioxidant capacity of pure phenolic compounds as well as of a phenolic rich extract, while also considering their possible pro-oxidant effects. The result obtained demonstrated that the proposed method allowed for the evaluation of the protection of DNA oxidation [in the presence of hydrogen peroxide (H 2 O 2 ) and an H 2 O 2 /iron (III) chloride (FeCl 3 ) systems] as well as for the observation of pro-oxidant activities, with the measurements registering interclass correlation coefficients above 0.9. Moreover, this method allowed for the characterization of the antioxidant capacity of a blueberry extract while demonstrating that it had no perceived pro-oxidant effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spectroscopic Tools for Quantitative Studies of DNA Structure and Dynamics

DEFF Research Database (Denmark)

Preus, Søren

The main objective of this thesis is to develop quantitative fluorescence-based, spectroscopic tools for probing the 3D structure and dynamics of DNA and RNA. The thesis is founded on six peer-reviewed papers covering mainly the development, characterization and use of fluorescent nucleobase...... analogues. In addition, four software packages is presented for the simulation and quantitative analysis of time-resolved and steady-state UV-Vis absorption and fluorescence experiments....

Modulation of mutagen-induced biological effects by inhibitors of DNA repair

International Nuclear Information System (INIS)

Natarajan, A.T.; Mullenders, L.F.H.; Zwanenburg, T.S.B.

1986-01-01

When lesions are induced in the DNA by mutagenic agents, they are subjected to cellular repair. Unrepaired and misrepaired lesions lead to biological effects, such as cell killing, point mutations and chromosomal alterations (aberrations and sister chromatid exchanges - SCEs). It is very difficult to directly correlate any particular type of lesion to a specific biological effect. However, in specific cases, this has been done. For example, short wave UV induced biological effects (cell killing, chromosomal alterations) result predominantly from induced cyclobutane dimers and by photoreactivation experiments, one can demonstrate that with the removal of dimers all types biological effects are diminished. In cases where many types of lesions are considered responsible for the observed biological effects other strategies have been employed to identify the possible lesion. The frequencies of induced chromosomal alterations and point mutations increase with the dose of the mutagen employed and an inhibition of DNA repair following treatment with the mutagen. Prevention of the cells from dividing following mutagen treatment allows them to repair premutational damage, thus reducing the biological effects induced. By comprehensive studies involving quantification of primary DNA lesions, their repair and biological effects will enable us to understand to some extent the complex processes involved in the manifestation of specific biological effects that follow the treatment of cells with mutagenic carcinogens

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

Science.gov (United States)

Lyko, Frank; Stach, Dirk; Brenner, Axel; Stilgenbauer, Stephan; Döhner, Hartmut; Wirtz, Michaela; Wiessler, Manfred; Schmitz, Oliver J

2004-06-01

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

Science.gov (United States)

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Heavy ion induced DNA transfer in biological cells

International Nuclear Information System (INIS)

Vilaithong, T.; Yu, L.D.; Apavatjrut, P.; Phanchaisri, B.; Sangyuenyongpipat, S.; Anuntalabhochai, S.; Brown, I.G.

2004-01-01

Low-energy ion beam bombardment of biological materials for genetic modification purposes has experienced rapid growth in the last decade, particularly for the direct DNA transfer into living organisms including both plants and bacteria. Attempts have been made to understand the mechanisms involved in ion-bombardment-induced direct gene transfer into biological cells. Here we summarize the present status of the application of low-energy ions for genetic modification of living sample materials

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

International Nuclear Information System (INIS)

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)

Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

Science.gov (United States)

Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

2008-12-15

Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines

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...... 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...... 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 100μm range. (Some...

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. © 2016 K. Hoffman, S. Leupen, et al. CBE—Life Sciences Education © 2016 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://creativecommons.org/licenses/by-nc-sa/3.0).

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.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

Science.gov (United States)

Ogino, S; Cantor, M; Kawasaki, T; Brahmandam, M; Kirkner, G J; Weisenberger, D J; Campan, M; Laird, P W; Loda, M; Fuchs, C S

2006-07-01

The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, pCIMP MSS tumours (6.6%, pCIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

Concentrations of environmental DNA (eDNA) reflect spawning salmon abundance at fine spatial and temporal scales

Science.gov (United States)

Tillotson, Michael D.; Kelly, Ryan P.; Duda, Jeff; Hoy, Marshal S.; Kralj, James; Quinn, Thomas P.

2018-01-01

Developing fast, cost-effective assessments of wild animal abundance is an important goal for many researchers, and environmental DNA (eDNA) holds much promise for this purpose. However, the quantitative relationship between species abundance and the amount of DNA present in the environment is likely to vary substantially among taxa and with ecological context. Here, we report a strong quantitative relationship between eDNA concentration and the abundance of spawning sockeye salmon in a small stream in Alaska, USA, where we took temporally- and spatially-replicated samples during the spawning period. This high-resolution dataset suggests that (1) eDNA concentrations vary significantly day-to-day, and likely within hours, in the context of the dynamic biological event of a salmon spawning season; (2) eDNA, as detected by species-specific quantitative PCR probes, seems to be conserved over short distances (tens of meters) in running water, but degrade quickly over larger scales (ca. 1.5 km); and (3) factors other than the mere presence of live, individual fish — such as location within the stream, live/dead ratio, and water temperature — can affect the eDNA-biomass correlation in space or time. A multivariate model incorporating both biotic and abiotic variables accounted for over 75% of the eDNA variance observed, suggesting that where a system is well-characterized, it may be possible to predict species' abundance from eDNA surveys, although we underscore that species- and system-specific variables are likely to limit the generality of any given quantitative model. Nevertheless, these findings provide an important step toward quantitative applications of eDNA in conservation and management.

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.

A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

African Journals Online (AJOL)

Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

A method for three-dimensional quantitative observation of the microstructure of biological samples

Science.gov (United States)

Wang, Pengfei; Chen, Dieyan; Ma, Wanyun; Wu, Hongxin; Ji, Liang; Sun, Jialin; Lv, Danyu; Zhang, Lu; Li, Ying; Tian, Ning; Zheng, Jinggao; Zhao, Fengying

2009-07-01

Contemporary biology has developed into the era of cell biology and molecular biology, and people try to study the mechanism of all kinds of biological phenomena at the microcosmic level now. Accurate description of the microstructure of biological samples is exigent need from many biomedical experiments. This paper introduces a method for 3-dimensional quantitative observation on the microstructure of vital biological samples based on two photon laser scanning microscopy (TPLSM). TPLSM is a novel kind of fluorescence microscopy, which has excellence in its low optical damage, high resolution, deep penetration depth and suitability for 3-dimensional (3D) imaging. Fluorescent stained samples were observed by TPLSM, and afterward the original shapes of them were obtained through 3D image reconstruction. The spatial distribution of all objects in samples as well as their volumes could be derived by image segmentation and mathematic calculation. Thus the 3-dimensionally and quantitatively depicted microstructure of the samples was finally derived. We applied this method to quantitative analysis of the spatial distribution of chromosomes in meiotic mouse oocytes at metaphase, and wonderful results came out last.

The yield, processing, and biological consequences of clustered DNA damage induced by ionizing radiation

International Nuclear Information System (INIS)

Shikazono, Naoya; Noguchi, Miho; Fujii, Kentaro; Urushibara, Ayumi; Yokoya, Akinari

2009-01-01

After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, chromosome aberration. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage. (author)

Opportunities for measuring DNA synthesis time by quantitative autoradiography

International Nuclear Information System (INIS)

Vasileva, D.

1980-01-01

DNA sysntesis time (Tsub(s)) in cells of the canine erythropoiesis and myelopoiesis pools was determined by quantitative autoradiography according to Doermer. In contrast to mitosis labelling for Tsub(s) estimation as so far applied, this technique uses well-differentiated cells. After blocking endogeneous DNA synthesis with 5-fluorodeoxyuridine, its further course becomes dependent on exogeneous supply of thymidine, in the form of 14 C-thymidine. From incroporation of the latter into the individual cell within a definite time span (3-7 min) and taking into account its total amount, Tsub(s) may be calculated. The data thus obtained were found to agree with Tsub(s) values as estimated from the labelled mitosis curve

Electrolytic reduction of nitroheterocyclic drugs leads to biologically important damage in DNA

International Nuclear Information System (INIS)

Lafleur, M.V.M.; Pluijmackers-Westmijze, E.J.; Loman, H.

1985-01-01

The effects of electrolytic reduction of nitroimidazole drugs on biologically active DNA was studied. The results show that reduction of the drugs in the presence of DNA affects inactivation for both double-stranded (RF) and single-stranded phiX174 DNA. However, stable reduction products did not make a significant contribution to the lethal damage in DNA. This suggests that probably a short-lived intermediate of reduction of nitro-compounds is responsible for damage to DNA. (author)

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.

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

International Nuclear Information System (INIS)

Dowd, D.R.; Lloyd, R.S.

1990-01-01

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance

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.

Amines as extracting agents for the quantitative determinations of actinides in biological samples

International Nuclear Information System (INIS)

Singh, N.P.

1987-01-01

The use of amines (primary, secondary and tertiary chains and quaternary ammonium salts) as extracting agents for the quantitative determination of actinides in biological samples is reviewed. Among the primary amines, only Primene JM-T is used to determine Pu in urine and bone. No one has investigated the possibility of using secondary amines to quantitatively extract actinides from biological samples. Among the tertiary amines, tri-n-octylamine, tri-iso-octylamine, tyricaprylamine (Alamine) and trilaurylamine (tridodecylamine) are used extensively to extract and separate the actinides from biological samples. Only one quaternary ammonium salt, methyltricapryl ammonium chloride (Aliquat-336), is used to extract Pu from biological samples. (author) 28 refs

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

Science.gov (United States)

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

Science.gov (United States)

Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

2015-07-01

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Quantitative measures of healthy aging and biological age

Science.gov (United States)

Kim, Sangkyu; Jazwinski, S. Michal

2015-01-01

Numerous genetic and non-genetic factors contribute to aging. To facilitate the study of these factors, various descriptors of biological aging, including ‘successful aging’ and ‘frailty’, have been put forth as integrative functional measures of aging. A separate but related quantitative approach is the ‘frailty index’, which has been operationalized and frequently used. Various frailty indices have been constructed. Although based on different numbers and types of health variables, frailty indices possess several common properties that make them useful across different studies. We have been using a frailty index termed FI34 based on 34 health variables. Like other frailty indices, FI34 increases non-linearly with advancing age and is a better indicator of biological aging than chronological age. FI34 has a substantial genetic basis. Using FI34, we found elevated levels of resting metabolic rate linked to declining health in nonagenarians. Using FI34 as a quantitative phenotype, we have also found a genomic region on chromosome 12 that is associated with healthy aging and longevity. PMID:26005669

Detection of Colorectal Cancer by a Quantitative Fluorescence Determination of DNA Amplification in Stool

Directory of Open Access Journals (Sweden)

Daniele Calistri

2004-09-01

Full Text Available DNA amplification of exfoliated cells in stool repre sents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, calle( fluorescence long DNA, was developed and validate( in our laboratory on stool obtained from 86 patient., with primary colorectal cancer and from 62 health individuals. It consists of the amplification of stoo DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of thi new method compared to the conventional approact was analyzed in a subgroup of 94 individuals (51 patients and 38 healthy volunteers. In the presen series, DNA amplification analysis showed a specific ity of 97% and a sensitivity of only 50%. Conversely fluorescence DNA evaluation, using the best cutoff o 25 ng, showed a sensitivity of about 76% and a spec ificity of 93%. Similar sensitivity was observed regard less of Dukes stage, tumor location, and size, thu., also permitting the detection of early-stage tumors The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.

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.

Convergence of The Nobel Fields of Telomere Biology and DNA Repair.

Science.gov (United States)

Fouquerel, Elise; Opresko, Patricia L

2017-01-01

The fields of telomere biology and DNA repair have enjoyed a great deal of cross-fertilization and convergence in recent years. Telomeres function at chromosome ends to prevent them from being falsely recognized as chromosome breaks by the DNA damage response and repair machineries. Conversely, both canonical and nonconical functions of numerous DNA repair proteins have been found to be critical for preserving telomere structure and function. In 2009, Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the Nobel prize in Physiology or Medicine for the discovery of telomeres and telomerase. Four years later, pioneers in the field of DNA repair, Aziz Sancar, Tomas Lindahl and Paul Modrich were recognized for their seminal contributions by being awarded the Nobel Prize in Chemistry. This review is part of a special issue meant to celebrate this amazing achievement, and will focus in particular on the convergence of nucleotide excision repair and telomere biology, and will discuss the profound implications for human health. © 2016 The American Society of Photobiology.

Quantitative live imaging of endogenous DNA replication in mammalian cells.

Directory of Open Access Journals (Sweden)

Andrew Burgess

Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

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....

A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

Science.gov (United States)

Sung, Myong-Hee

2013-01-01

Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. PMID:24709701

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

Science.gov (United States)

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

2011-01-01

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

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...

Radiation-induced DNA-protein cross-links: Mechanisms and biological significance.

Science.gov (United States)

Nakano, Toshiaki; Xu, Xu; Salem, Amir M H; Shoulkamy, Mahmoud I; Ide, Hiroshi

2017-06-01

Ionizing radiation produces various DNA lesions such as base damage, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, the biological significance of DPCs remains elusive. In this article, we focus on radiation-induced DPCs and review the current understanding of their induction, properties, repair, and biological consequences. When cells are irradiated, the formation of base damage, SSBs, and DSBs are promoted in the presence of oxygen. Conversely, that of DPCs is promoted in the absence of oxygen, suggesting their importance in hypoxic cells, such as those present in tumors. DNA and protein radicals generated by hydroxyl radicals (i.e., indirect effect) are responsible for DPC formation. In addition, DPCs can also be formed from guanine radical cations generated by the direct effect. Actin, histones, and other proteins have been identified as cross-linked proteins. Also, covalent linkages between DNA and protein constituents such as thymine-lysine and guanine-lysine have been identified and their structures are proposed. In irradiated cells and tissues, DPCs are repaired in a biphasic manner, consisting of fast and slow components. The half-time for the fast component is 20min-2h and that for the slow component is 2-70h. Notably, radiation-induced DPCs are repaired more slowly than DSBs. Homologous recombination plays a pivotal role in the repair of radiation-induced DPCs as well as DSBs. Recently, a novel mechanism of DPC repair mediated by a DPC protease was reported, wherein the resulting DNA-peptide cross-links were bypassed by translesion synthesis. The replication and transcription of DPC-bearing reporter plasmids are inhibited in cells, suggesting that DPCs are potentially lethal lesions. However, whether DPCs are mutagenic and induce gross chromosomal alterations remains to be determined. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

Science.gov (United States)

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

A biologically inspired two-species exclusion model: effects of RNA polymerase motor traffic on simultaneous DNA replication

Science.gov (United States)

Ghosh, Soumendu; Mishra, Bhavya; Patra, Shubhadeep; Schadschneider, Andreas; Chowdhury, Debashish

2018-04-01

We introduce a two-species exclusion model to describe the key features of the conflict between the RNA polymerase (RNAP) motor traffic, engaged in the transcription of a segment of DNA, concomitant with the progress of two DNA replication forks on the same DNA segment. One of the species of particles (P) represents RNAP motors while the other (R) represents the replication forks. Motivated by the biological phenomena that this model is intended to capture, a maximum of two R particles only are allowed to enter the lattice from two opposite ends whereas the unrestricted number of P particles constitutes a totally asymmetric simple exclusion process (TASEP) in a segment in the middle of the lattice. The model captures three distinct pathways for resolving the co-directional as well as head-on collision between the P and R particles. Using Monte Carlo simulations and heuristic analytical arguments that combine exact results for the TASEP with mean-field approximations, we predict the possible outcomes of the conflict between the traffic of RNAP motors (P particles engaged in transcription) and the replication forks (R particles). In principle, the model can be adapted to experimental conditions to account for the data quantitatively.

Biological characteristics of crucian by quantitative inspection method

Science.gov (United States)

Chu, Mengqi

2015-04-01

Biological characteristics of crucian by quantitative inspection method Through quantitative inspection method , the biological characteristics of crucian was preliminary researched. Crucian , Belongs to Cypriniformes, Cyprinidae, Carassius auratus, is a kind of main plant-eating omnivorous fish,like Gregarious, selection and ranking. Crucian are widely distributed, perennial water all over the country all have production. Determine the indicators of crucian in the experiment, to understand the growth, reproduction situation of crucian in this area . Using the measured data (such as the scale length ,scale size and wheel diameter and so on) and related functional to calculate growth of crucian in any one year.According to the egg shape, color, weight ,etc to determine its maturity, with the mean egg diameter per 20 eggs and the number of eggs per 0.5 grams, to calculate the relative and absolute fecundity of the fish .Measured crucian were female puberty. Based on the relation between the scale diameter and length and the information, linear relationship between crucian scale diameter and length: y=1.530+3.0649. From the data, the fertility and is closely relative to the increase of age. The older, the more mature gonad development. The more amount of eggs. In addition, absolute fecundity increases with the pituitary gland.Through quantitative check crucian bait food intake by the object, reveals the main food, secondary foods, and chance food of crucian ,and understand that crucian degree of be fond of of all kinds of bait organisms.Fish fertility with weight gain, it has the characteristics of species and populations, and at the same tmes influenced by the age of the individual, body length, body weight, environmental conditions (especially the nutrition conditions), and breeding habits, spawning times factors and the size of the egg. After a series of studies of crucian biological character, provide the ecological basis for local crucian's feeding, breeding

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study

NARCIS (Netherlands)

Hammond, Emma L.; Sayer, David; Nolan, David; Walker, Ulrich A.; Ronde, Anthony de; Montaner, Julio S. G.; Cote, Helene C. F.; Gahan, Michelle E.; Cherry, Catherine L.; Wesselingh, Steven L.; Reiss, Peter; Mallal, Simon

2003-01-01

Background: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. Objectives: A collaborative study was undertaken to evaluate intra-assay precision and between

Theory and Practice in Quantitative Genetics

DEFF Research Database (Denmark)

Posthuma, Daniëlle; Beem, A Leo; de Geus, Eco J C

2003-01-01

With the rapid advances in molecular biology, the near completion of the human genome, the development of appropriate statistical genetic methods and the availability of the necessary computing power, the identification of quantitative trait loci has now become a realistic prospect for quantitative...... geneticists. We briefly describe the theoretical biometrical foundations underlying quantitative genetics. These theoretical underpinnings are translated into mathematical equations that allow the assessment of the contribution of observed (using DNA samples) and unobserved (using known genetic relationships......) genetic variation to population variance in quantitative traits. Several statistical models for quantitative genetic analyses are described, such as models for the classical twin design, multivariate and longitudinal genetic analyses, extended twin analyses, and linkage and association analyses. For each...

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

Energy Technology Data Exchange (ETDEWEB)

LaRiviere, Frederick J. [Department of Chemistry, Washington and Lee University, Lexington, VA 24450 (United States); Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E. [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States); Greenwood, Paul G. [Department of Biology, Colby College, Waterville, ME 04901 (United States); Millard, Julie T., E-mail: jtmillar@colby.edu [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States)

2009-05-12

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

International Nuclear Information System (INIS)

LaRiviere, Frederick J.; Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E.; Greenwood, Paul G.; Millard, Julie T.

2009-01-01

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Effects of formic acid hydrolysis on the quantitative analysis of radiation-induced DNA base damage products assayed by gas chromatography/mass spectrometry

International Nuclear Information System (INIS)

Swarts, S.G.; Smith, G.S.; Miao, L.; Wheeler, K.T.

1996-01-01

Gas chromatography/mass spectrometry (GC/ MS-SIM) is an excellent technique for performing both qualitative and quantitative analysis of DNA base damage products that are formed by exposure to ionizing radiation or by the interaction of intracellular DNA with activated oxygen species. This technique commonly uses a hot formic acid hydrolysis step to degrade the DNA to individual free bases. However, due to the harsh nature of this degradation procedure, the quantitation of DNA base damage products may be adversely affected. Consequently, we examined the effects of various formic acid hydrolysis procedures on the quantitation of a number of DNA base damage products and identified several factors that can influence this quantitation. These factors included (1) the inherent acid stabilities of both the lesions and the internal standards; (2) the hydrolysis temperature; (3) the source and grade of the formic acid; and (4) the sample mass during hydrolysis. Our data also suggested that the N, O-bis (trimethylsilyl)trifluoroacetamide (BSTFA) derivatization efficiency can be adversely affected, presumably by trace contaminants either in the formic acid or from the acid-activated surface of the glass derivatization vials. Where adverse effects were noted, modifications were explored in an attempt to improve the quantitation of these DNA lesions. Although experimental steps could be taken to minimize the influence of these factors on the quantitation of some base damage products, no single procedure solved the quantitation problem for all base lesions. However, a significant improvement in the quantitation was achieved if the relative molecular response factor (RMRF) values for these lesions were generated with authentic DNA base damage products that had been treated exactly like the experimental samples. (orig.)

Nuclear physics and biology

International Nuclear Information System (INIS)

Valentin, L.

1994-01-01

This paper is about nuclear instrumentation and biological concepts, based on images from appropriate Β detectors. First, three detectors are described: the SOFI detector, for gene mapping, the SOFAS detector, for DNA sequencing and the RIHR detector, for in situ hybridization. Then, the paper presents quantitative imaging in molecular genetic and functional imaging. (TEC)

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

Science.gov (United States)

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Quantitative strategies to determine cisplatin adducts with DNA nucleotides in drosofila larvae and tumoral cell cultures

International Nuclear Information System (INIS)

Garcia Sar, D.; Montes-Bayon, M.; Hann, S.; Koellensperger, G.; Blanco-Gonzalez, E.; Sanz-Medel, A.

2009-01-01

Full text: The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. A novel sensitive and selective method is proposed to quantify cisplatin-DNA adducts induced in vivo in somatic cells of Drosophila melanogaster at biologically relevant concentrations. The method uses HPLC-ICPMS in combination with species-specific isotope dilution analysis (cisplatin enriched in 194 Pt). For the first time, a cisplatin-DNA adduct is quantified by this approach. The obtained results show the great potential of this system to advance our molecular understanding of the biological effects of cisplatin. (author)

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

Science.gov (United States)

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

Science.gov (United States)

van Dolleweerd, Craig J; Kessans, Sarah A; Van de Bittner, Kyle C; Bustamante, Leyla Y; Bundela, Rudranuj; Scott, Barry; Nicholson, Matthew J; Parker, Emily J

2018-04-20

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

Analysis of DNA interactions using single-molecule force spectroscopy.

Science.gov (United States)

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

2013-06-01

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Biphasic dose responses in biology, toxicology and medicine: Accounting for their generalizability and quantitative features

International Nuclear Information System (INIS)

Calabrese, Edward J.

2013-01-01

The most common quantitative feature of the hormetic-biphasic dose response is its modest stimulatory response which at maximum is only 30–60% greater than control values, an observation that is consistently independent of biological model, level of organization (i.e., cell, organ or individual), endpoint measured, chemical/physical agent studied, or mechanism. This quantitative feature suggests an underlying “upstream” mechanism common across biological systems, therefore basic and general. Hormetic dose response relationships represent an estimate of the peak performance of integrative biological processes that are allometrically based. Hormetic responses reflect both direct stimulatory or overcompensation responses to damage induced by relatively low doses of chemical or physical agents. The integration of the hormetic dose response within an allometric framework provides, for the first time, an explanation for both the generality and the quantitative features of the hormetic dose response. -- Highlights: •The hormetic stimulation is at maximum 30–60% greater than control responses. •Hormesis is a measure of biological performance and plasticity. •The hormetic response is evolutionary based and highly generalizable. -- This paper provides a biologically based explanation for the generalizability/quantitative features of the hormetic dose response, representing a fundamental contribution to the field

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'.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

Science.gov (United States)

Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

2014-11-01

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

Science.gov (United States)

Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

2016-12-02

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

International Nuclear Information System (INIS)

Cox, Annie M.; Goodwin, Kelly D.

2013-01-01

Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

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.

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. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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

International Nuclear Information System (INIS)

Li Mengnan; Zheng Guanghong; Wang Lei; Xiao Wei; Fu Xiaohua; Le Yiquan; Ren Daming

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 deg. 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 deg. 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 deg. 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

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.

2015-01-01

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. PMID:25155200

DNA markers for forensic identification of non-human biological traces

NARCIS (Netherlands)

Wesselink, M.

2018-01-01

In this thesis, DNA markers are described that enable forensically relevant classification of three groups of non-human biological traces: fungi (Chapter 1), domestic cats (Chapters 2, 3 an d 4) and birch trees (Chapters 5 and 6). Because the forensic questions associated with these traces require

An immunochemical approach to the study of DNA damage and repair

International Nuclear Information System (INIS)

Wallace, S.S.; Erlanger, B.F.

1992-05-01

The overall objective of this project has been to develop immunochemical methods to quantitate unique DNA base damages in order to facilitate studies on radiation-induced damage production and repair. Specifically, we have been using antibodies raised to damaged bases to quantitate unique lesions in model systems in order to evaluate their potential biological consequences. Our approach has been to synthesize modified nucleotides or nucleosides, conjugate them to protein carriers, and use the conjugates as immunogens in rabbits or to prepare monoclonal antibodies. We have been studying damages that are stable radiolysis products found in X-irradiated DNA and thus of potential biological consequence. Our aim is to build an in vitro and in vivo data base on the interactions between model DNA lesions and such cellular enzymes as DNA polymerases and repair endonucleases. Initial studies have focused on pyrimidine ring saturation products (thymine glycol.and dihydrothymine), products resulting from ring fragmentation or base loss (urea, Β-ureidoisobutyric acid, abasic sites), 7-hydro-8-oxopurines, and more recently, cytosine radiolysis products. These modified bases serve as useful models for examining the potential lethal and/or mutagenic (carcinogenic) effects of the products of DNA radiolysis

A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks.

Science.gov (United States)

Pachkowski, Brian; Nakamura, Jun

2013-01-01

Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.

Effect of oxygen on inactivation of biologically active DNA by γ rays in vitro: influence of metalloporphyrins and enzymatic DNA repair

International Nuclear Information System (INIS)

van Hemmen, J.J.; Meuling, W.J.A.; Bleichrodt, J.F.

1978-01-01

Biologically active DNA dissolved in a bacterial extract shows a higher sensitivity to γ rays under oxygen than under anoxic conditions. This oxygen effect depends on the presence of dialyzable, probably organometallic, compounds in the extract. Metalloporphyrins mimic these cellular components with regard to the effect of oxygen on DNA irradiated in vitro. Anoxic irradiation leads to less double-strand breaks in the DNA than irradiation under oxygen, but the oxygen effect in vitro is mainly due to nucleotide damage. No oxygen effect is observed when the biological activity of the irradiated DNA is assayed on spheroplasts of a bacterial strain carrying a uvrA mutation, i.e., a deficiency in the excision repair system, and the sensitivity of the DNA is almost equal to that found for irradiation under oxygen and assay on a repair-proficient strain. It may be concluded, therefore, that the oxygen effect observed with DNA in cellular extracts or in the presence of metalloporphyrins results from more efficient cellular repair of the otherwise lethal nucleotide damage inflicted under anoxic conditions. Comparison of the oxygen effect on DNA in vitro with the radiosensitization of bacterial cells by oxygen shows that in bacteria part of the radiation damage may be similar to that induced in DNA in vitro, but, in addition, the cells sustain another type of damage which is subjected to an oxygen effect but not to excision repair

Influence of anoxic sensitizers on the radiation damage in biologically active DNA in aqueous solution

International Nuclear Information System (INIS)

Lafleur, M.V.M.; Loman, H.

1982-01-01

The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime. (author)

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.

Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

DEFF Research Database (Denmark)

Fazio, Alessandro

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...... 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...

Quantitative Visualization of ChIP-chip Data by Using Linked Views

Energy Technology Data Exchange (ETDEWEB)

Huang, Min-Yu; Weber, Gunther; Li, Xiao-Yong; Biggin, Mark; Hamann, Bernd

2010-11-05

Most analyses of ChIP-chip in vivo DNA binding have focused on qualitative descriptions of whether genomic regions are bound or not. There is increasing evidence, however, that factors bind in a highly overlapping manner to the same genomic regions and that it is quantitative differences in occupancy on these commonly bound regions that are the critical determinants of the different biological specificity of factors. As a result, it is critical to have a tool to facilitate the quantitative visualization of differences between transcription factors and the genomic regions they bind to understand each factor's unique roles in the network. We have developed a framework which combines several visualizations via brushing-and-linking to allow the user to interactively analyze and explore in vivo DNA binding data of multiple transcription factors. We describe these visualization types and also provide a discussion of biological examples in this paper.

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

International Nuclear Information System (INIS)

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)

A nuclear DNA-based species determination and DNA quantification assay for common poultry species.

Science.gov (United States)

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

2014-12-01

DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

Quantitative assessment of biological impact using transcriptomic data and mechanistic network models

International Nuclear Information System (INIS)

Thomson, Ty M.; Sewer, Alain; Martin, Florian; Belcastro, Vincenzo; Frushour, Brian P.; Gebel, Stephan; Park, Jennifer; Schlage, Walter K.; Talikka, Marja; Vasilyev, Dmitry M.; Westra, Jurjen W.; Hoeng, Julia; Peitsch, Manuel C.

2013-01-01

Exposure to biologically active substances such as therapeutic drugs or environmental toxicants can impact biological systems at various levels, affecting individual molecules, signaling pathways, and overall cellular processes. The ability to derive mechanistic insights from the resulting system responses requires the integration of experimental measures with a priori knowledge about the system and the interacting molecules therein. We developed a novel systems biology-based methodology that leverages mechanistic network models and transcriptomic data to quantitatively assess the biological impact of exposures to active substances. Hierarchically organized network models were first constructed to provide a coherent framework for investigating the impact of exposures at the molecular, pathway and process levels. We then validated our methodology using novel and previously published experiments. For both in vitro systems with simple exposure and in vivo systems with complex exposures, our methodology was able to recapitulate known biological responses matching expected or measured phenotypes. In addition, the quantitative results were in agreement with experimental endpoint data for many of the mechanistic effects that were assessed, providing further objective confirmation of the approach. We conclude that our methodology evaluates the biological impact of exposures in an objective, systematic, and quantifiable manner, enabling the computation of a systems-wide and pan-mechanistic biological impact measure for a given active substance or mixture. Our results suggest that various fields of human disease research, from drug development to consumer product testing and environmental impact analysis, could benefit from using this methodology. - Highlights: • The impact of biologically active substances is quantified at multiple levels. • The systems-level impact integrates the perturbations of individual networks. • The networks capture the relationships between

On the analysis of complex biological supply chains: From Process Systems Engineering to Quantitative Systems Pharmacology.

Science.gov (United States)

Rao, Rohit T; Scherholz, Megerle L; Hartmanshenn, Clara; Bae, Seul-A; Androulakis, Ioannis P

2017-12-05

The use of models in biology has become particularly relevant as it enables investigators to develop a mechanistic framework for understanding the operating principles of living systems as well as in quantitatively predicting their response to both pathological perturbations and pharmacological interventions. This application has resulted in a synergistic convergence of systems biology and pharmacokinetic-pharmacodynamic modeling techniques that has led to the emergence of quantitative systems pharmacology (QSP). In this review, we discuss how the foundational principles of chemical process systems engineering inform the progressive development of more physiologically-based systems biology models.

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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.

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.

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

International Nuclear Information System (INIS)

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

2014-01-01

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. - 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

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.

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.

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

Directory of Open Access Journals (Sweden)

Matthias Christen

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

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

Science.gov (United States)

Thomas, Marcus; Schwartz, Russell

2017-05-23

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.

The current status of studies on mitochondrial DNA with tumor, radiation biological effects and aging

International Nuclear Information System (INIS)

Liu Qingjie; Sang Lu

2004-01-01

The mitochondrial plays a very important role in sustaining the normal physiological function, because it is the center of energy making and mitochondrial DNA (mtDNA) is the only genetic material outside the nuclear. The result of studies showed that many diseases have a close relationship with mtDNA mutation and deletion. This article reviewed the current status of research on mtDNA with tumor, radiation biological effects and aging, in order to initiate the application study of mtDNA in the circle of radiation medicine

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)

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

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.

Stochastic filtering of quantitative data from STR DNA analysis

DEFF Research Database (Denmark)

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

due to the apparatus used for measurements). Pull-up effects (more systematic increase caused by overlap in the spectrum) Stutters (peaks located four basepairs before the true peak). We present filtering techniques for all three technical artifacts based on statistical analysis of data from......The quantitative data observed from analysing STR DNA is a mixture of contributions from various sources. Apart from the true allelic peaks, the observed signal consists of at least three components resulting from the measurement technique and the PCR amplification: Background noise (random noise...... controlled experiments conducted at The Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, Universityof Copenhagen, Denmark....

Study on methods of quantitative analysis of the biological thin samples in EM X-ray microanalysis

International Nuclear Information System (INIS)

Zhang Detian; Zhang Xuemin; He Kun; Yang Yi; Zhang Sa; Wang Baozhen

2000-01-01

Objective: To study the methods of quantitative analysis of the biological thin samples. Methods: Hall theory was used to study the qualitative analysis, background subtraction, peel off overlap peaks; external radiation and aberrance of spectra. Results: The results of reliable qualitative analysis and precise quantitative analysis were achieved. Conclusion: The methods for analysis of the biological thin samples in EM X-ray microanalysis can be used in biomedical research

Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

International Nuclear Information System (INIS)

Dambergs, R.; Kidson, C.

1979-01-01

Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

Development of 19F-NMR chemical shift detection of DNA B-Z equilibrium using 19F-NMR.

Science.gov (United States)

Nakamura, S; Yang, H; Hirata, C; Kersaudy, F; Fujimoto, K

2017-06-28

Various DNA conformational changes are in correlation with biological events. In particular, DNA B-Z equilibrium showed a high correlation with translation and transcription. In this study, we developed a DNA probe containing 5-trifluoromethylcytidine or 5-trifluoromethylthymidine to detect DNA B-Z equilibrium using 19 F-NMR. Its probe enabled the quantitative detection of B-, Z-, and ss-DNA based on 19 F-NMR chemical shift change.

A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

Science.gov (United States)

Kafle, Amol; Klaene, Joshua; Hall, Adam B; Glick, James; Coy, Stephen L; Vouros, Paul

2013-07-15

There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis. A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP). After optimization of the DMS parameters, each sample was analyzed in just 30 s following a simple protein precipitation step of the digested DNA. A detection limit of one modification in 10^6 nucleosides has been achieved using only 2 µg of DNA. A brief comparison (quantitative and qualitative) with liquid chromatography/mass spectrometry is also presented highlighting the advantages of using the DMS/MS method as a high-throughput platform. The data presented demonstrate the successful application of a DMS/MS/MS platform for the rapid quantitation of DNA adducts using, as a model analyte, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl. Copyright © 2013 John Wiley & Sons, Ltd.

Microfluorometric mithramycin assay for quantitating the effects of immunotoxicants on lymphocyte activation

International Nuclear Information System (INIS)

Quattrone, A.J.; Ranney, D.F.

1981-01-01

A semiautomated, microfluorometric assay has been developed for the detection of toxicant-induced changes in lymphocyte DNA content at standard intervals after mitogen activation. DNA is quantitated by solubilizing the cells and determining the fluorescence enhancement that results from formation of the highly specific mithramycin:DNA adduct. The limit of detection is 0.21 μg (30,000 resting cell equivalents) per microliter well. Correlation with the less sensitive, nonautomatable, diphenylamine DNA assay give a correlation coefficient r = 0.91. Prototype substances representative of true immunotoxicants (prostaglandin E 2 ) and common interfering substances (thymidine at 14 M) have been tested. The latter substance produces false positive results in the standard [ 3 H] thymidine assay. The mithramycin assay does not inappropriately detect this interfering substance. It has the characteristics of a highly specific, accurate technique of screening and quantitating immunotoxic drugs, agents, and mediators in patient sera and other complex biological fluids

RAFT-mediated synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride] brushes for quantitative DNA immobilization

International Nuclear Information System (INIS)

Demirci, Serkan; Caykara, Tuncer

2013-01-01

The synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride)] [poly(VBTAC)] brushes was achieved via reversible addition-fragmentation chain transfer (RAFT) polymerization and used for quantitative DNA immobilization. Initially, silicon surfaces were modified with RAFT chain transfer agent by utilizing an amide reaction involving a silicon wafer modified with allylamine and 4-cyanopentanoic acid dithiobenzoate (CPAD). Poly(VBTAC) brushes were then prepared via RAFT-mediated polymerization from the surface immobilized CPAD. Various characterization techniques including ellipsometry, X-ray photoelectron spectroscopy, grazing angle-Fourier transform infrared spectroscopy, atomic force microscopy and contact-angle goniometer were used to characterize the immobilization of CPAD on the silicon wafer and the subsequent polymer formation. The addition of free CPAD was required for the formation of well-defined polymer brushes, which subsequently resulted in the presence of free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. Moreover, from atomic force microscopy and ellipsometry measurements, it was also determined that the density of immobilized DNA on the cationic poly(VBTAC) brushes can be quantitatively controlled by adjusting the solution concentration. Highlights: ► The cationic poly(VBTAC) brushes were prepared by RAFT polymerization. ► Grafting density of cationic poly(VBTAC) brushes was as high as 0.76 chains/nm 2 . ► The cationic poly(VBTAC) brushes were used for quantitative DNA immobilization.

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

Science.gov (United States)

Oberstrass, Florian C.; Fernandes, Louis E.; Bryant, Zev

2012-01-01

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. “Bubble” templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context. PMID:22474350

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

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

Science.gov (United States)

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.

Science.gov (United States)

Xiao, Lu; Xiang, Yu

2016-06-01

The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stress and DNA repair biology of the Fanconi anemia pathway

Science.gov (United States)

Longerich, Simonne; Li, Jian; Xiong, Yong; Sung, Patrick

2014-01-01

Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care. PMID:25237197

Qualitative and quantitative assessment of DNA quality of frozen beef based on DNA yield, gel electrophoresis and PCR amplification and their correlations to beef quality.

Science.gov (United States)

Zhao, Jing; Zhang, Ting; Liu, Yongfeng; Wang, Xingyu; Zhang, Lan; Ku, Ting; Quek, Siew Young

2018-09-15

Freezing is a practical method for meat preservation but the quality of frozen meat can deteriorate with storage time. This research investigated the effect of frozen storage time (up to 66 months) on changes in DNA yield, purity and integrity in beef, and further analyzed the correlation between beef quality (moisture content, protein content, TVB-N value and pH value) and DNA quality in an attempt to establish a reliable, high-throughput method for meat quality control. Results showed that frozen storage time influenced the yield and integrity of DNA significantly (p quality degraded dramatically with the increased storage time based on gel electrophoresis results. Polymerase chain reaction (PCR) products from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were observed in all frozen beef samples. Using real-time PCR for quantitative assessment of DNA and meat quality revealed that correlations could be established successfully with mathematical models to evaluate frozen beef quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

In vitro studies. Contribution of radioactive marking to molecular biology development

International Nuclear Information System (INIS)

Sentenac, A.

1997-01-01

The spectacular and rapid development of molecular biology is essentially related to the utilization of marked molecules which leads to quantitative and qualitative information; the use of radioactive tracers allowed for the observation of the biosynthesis of biological polymers, and thus, for example, the formation of DNA, RNA or proteins. A historical review of the great discoveries in this field, is presented

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

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.

Quantitation of the repair of gamma-radiation-induced double-strand DNA breaks in human fibroblasts

International Nuclear Information System (INIS)

Woods, W.G.

1981-01-01

The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a tsub(1/2) of 10 min for repair of 5000 rad exposure and 39 min for repair of 10000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial fast and a later slow mechanism may be involved. (Auth.)

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.

A biological inspired fuzzy adaptive window median filter (FAWMF) for enhancing DNA signal processing.

Science.gov (United States)

Ahmad, Muneer; Jung, Low Tan; Bhuiyan, Al-Amin

2017-10-01

Digital signal processing techniques commonly employ fixed length window filters to process the signal contents. DNA signals differ in characteristics from common digital signals since they carry nucleotides as contents. The nucleotides own genetic code context and fuzzy behaviors due to their special structure and order in DNA strand. Employing conventional fixed length window filters for DNA signal processing produce spectral leakage and hence results in signal noise. A biological context aware adaptive window filter is required to process the DNA signals. This paper introduces a biological inspired fuzzy adaptive window median filter (FAWMF) which computes the fuzzy membership strength of nucleotides in each slide of window and filters nucleotides based on median filtering with a combination of s-shaped and z-shaped filters. Since coding regions cause 3-base periodicity by an unbalanced nucleotides' distribution producing a relatively high bias for nucleotides' usage, such fundamental characteristic of nucleotides has been exploited in FAWMF to suppress the signal noise. Along with adaptive response of FAWMF, a strong correlation between median nucleotides and the Π shaped filter was observed which produced enhanced discrimination between coding and non-coding regions contrary to fixed length conventional window filters. The proposed FAWMF attains a significant enhancement in coding regions identification i.e. 40% to 125% as compared to other conventional window filters tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms. This study proves that conventional fixed length window filters applied to DNA signals do not achieve significant results since the nucleotides carry genetic code context. The proposed FAWMF algorithm is adaptive and outperforms significantly to process DNA signal contents. The algorithm applied to variety of DNA datasets produced noteworthy discrimination between coding and non-coding regions contrary

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...

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

Directory of Open Access Journals (Sweden)

Stefanie Fischer

Full Text Available The biological relevance of extracellular vesicles (EV in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

Science.gov (United States)

Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.ABSTRACTDNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

DNA Nucleotides Detection via capacitance properties of Graphene

Science.gov (United States)

Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash

2016-05-01

In the present paper a new method is suggested to detect the DNA nucleotides on a first-principles calculation of the electronic features of DNA bases which chemisorbed to a graphene sheet placed between two gold electrodes in a contact-channel-contact system. The capacitance properties of graphene in the channel are surveyed using non-equilibrium Green's function coupled with the Density Functional Theory. Thus, the capacitance properties of graphene are theoretically investigated in a biological environment, and, using a novel method, the effect of the chemisorbed DNA nucleotides on electrical charges on the surface of graphene is deciphered. Several parameters in this method are also extracted including Electrostatic energy, Induced density, induced electrostatic potential, Electron difference potential and Electron difference density. The qualitative and quantitative differences among these parameters can be used to identify DNA nucleotides. Some of the advantages of this approach include its ease and high accuracy. What distinguishes the current research is that it is the first experiment to investigate the capacitance properties of gaphene changes in the biological environment and the effect of chemisorbed DNA nucleotides on the surface of graphene on the charge.

DNA internal standard for the quantitative determination of hallucinogenic plants in plant mixtures.

Science.gov (United States)

Luciano, Pino; Bertea, Cinzia M; Temporale, Giovanni; Maffei, Massimo E

2007-12-01

Here, we show a new, simple, and rapid SYBR Green-based Real-Time PCR assay for the quantification of hallucinogenic plants in plant mixtures. As a test plant, Salvia divinorum Epling & Játiva-M., a perennial herb belonging to the Lamiaceae family able to induce hallucinations, changes in perception, or other psychologically induced changes with similar potency as LSD, was used. The method was tested on seven mixtures 100/0%, 80/20%, 60/40%, 40/60%, 20/80%, 10/90%, 0/100% (w/w) S. divinorum versus a non-hallucinogenic plant, Salvia officinalis. Total DNA was extracted from samples and quantified by Real-Time PCR. Arabidopsis thaliana genomic DNA was added, as internal standard, at the beginning of each extraction. A new formula for the interpretation of Real-Time PCR data, based on the relative quantification of DNA extracted from mixture versus a reference DNA extracted from a known amount of pure S. divinorum, was developed. The results of this work show an almost perfect correspondence between Real-Time PCR-calculated weight and the weight estimated by an analytical weighted method, proving the effectiveness of this method for the quantitative analysis of a given species in a plant mixture.

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

Science.gov (United States)

Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

2001-01-01

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

Scintillating Optical Fiber Imagers for biology

International Nuclear Information System (INIS)

Mastrippolito, R.

1990-01-01

S.O.F.I (Scintillating Optical Fiber Imager) is a detector developed to replace the autoradiographic films used in molecular biology for the location of radiolabelled ( 32 P) DNA molecules in blotting experiments. It analyses samples on a 25 x 25 cm 2 square area still 25 times faster than autoradiographic films, with a 1.75 and 3 mm resolution for two orthogonal directions. This device performs numerised images with a dynamic upper than 100 which allows the direct quantitation of the analysed samples. First, this thesis describes the S.O.F.I. development (Scintillating Optical Fibers, coding of these fibers and specific electronic for the treatment of the Multi-Anode Photo-Multiplier signals) and experiments made in collaboration with molecular biology laboratories. In a second place, we prove the feasibility of an automatic DNA sequencer issued from S.O.F.I [fr

Physico-chemical and biological study of excision-repair of UV-irradiated PHIX 174 RF DNA in vitro

International Nuclear Information System (INIS)

Heijneker, H.L.

1975-01-01

A study is presented on the excision repair of ultraviolet-irradiated PHIX 174 RFI DNA in vitro with UV-specific endonuclease from micrococcus luteus, DNA polymerase I from E. coli and DNA ligase from phage T 4 infected E. coli. Excision repair was measured by physico-chemical and by biological methods. It is shown that more than 90% of the pyrimidine dimers can be repaired in vitro and that the repaired molecules have regained full biological activity. Endonuclease III was not essential for excision repair in vitro and did not stimulate repair; from this it was concluded that UV-endo generates 3' OH endgroups. The usefulness of the methods with regard to the study of excision repair is discussed

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...

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....

Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

International Nuclear Information System (INIS)

Gruschus, James M.; Ferretti, James A.

2001-01-01

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant

“DNA in the time tunnel”: a report of extensionist activity for biology teaching

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Elison de Souza Sevalho

2017-06-01

Full Text Available This paper reports the experience of the extension project entitled “DNA in the time tunnel”. This project was developed with high school finalists students of a public school in the city of Coari, State of Amazonas, Brazil, aiming to provide students and teachers of biology and chemistry, teaching and learning about the historical context of the elucidation of DNA. The intervention was carried out in two stages: the first was the bibliographic research and planning and preparation of materials with playful bias, showing the contribution of each researcher and a gymkhana as an instrument to contribute to the learning of biology and the execution of extensionist activities with students and teachers. The project actions have contributed to the planning of the dynamic pedagogical practices, which granted the needs and interests of the involved students; to the enrichment of the knowledge on the subject addressed by secondary students, training them with matters of biology that are in the National Secondary Education Examination (ENEM and other selective processes of entry to higher education; to the teaching and learning of biological disciplines of the curriculum of the respective college freshmen courses of the Institute of health and biotechnology.

Investigation of the effect of ionizing radiation on gene expression variation by the 'DNA chips': feasibility of a biological dosimeter

International Nuclear Information System (INIS)

Gruel, G.

2005-01-01

After having described the different biological effects of ionizing radiation and the different approaches to biological dosimetry, and introduced 'DNA chips' or DNA micro-arrays, the author reports the characterization of gene expression variations in the response of cells to a gamma irradiation. Both main aspects of the use DNA chips are investigated: fundamental research and diagnosis. This research thesis thus proposes an analysis of the effect of ionizing radiation using DNA chips, notably by comparing gene expression modifications measured in mouse irradiated lung, heart and kidney. It reports a feasibility study of bio-dosimeter based on expression profiles

Quantitative phase imaging and differential interference contrast imaging for biological TEM

International Nuclear Information System (INIS)

Allman, B.E.; McMahon, P.J.; Barone-Nugent, E.D.; Nugent, E.D.

2002-01-01

Full text: Phase microscopy is a central technique in science. An experienced microscopist uses this effect to visualise (edge) structure within transparent samples by slightly defocusing the microscope. Although widespread in optical microscopy, phase contrast transmission electron microscopy (TEM) has not been widely adopted. TEM for biological specimens has largely relied on staining techniques to yield sufficient contrast. We show here a simple method for quantitative TEM phase microscopy that quantifies this phase contrast effect. Starting with conventional, digital, bright field images of the sample, our algorithm provides quantitative phase information independent of the sample's bright field intensity image. We present TEM phase images of a range of stained and unstained, biological and material science specimens. This independent phase and intensity information is then used to emulate a range of phase visualisation images familiar to optical microscopy, e.g. differential interference contrast. The phase images contain features not visible with the other imaging modalities. Further, if the TEM samples have been prepared on a microtome to a uniform thickness, the phase information can be converted into refractive index structure of the specimen. Copyright (2002) Australian Society for Electron Microscopy Inc

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.

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.

Molecular biological mechanisms I. DNA repair

International Nuclear Information System (INIS)

Friedl, A.A.

2000-01-01

Cells of all living systems possess a variety of mechanisms that allow to repair spontaneous and exogeneously induced DNA damage. DNA repair deficiencies may invoke enhanced sensitivity towards DNA-damaging agents such as ionizing radiation. They may also enhance the risk of cancer development, both spontaneously or after induction. This article reviews several DNA repair mechanisms, especially those dealing with DNA double-strand breaks, and describes hereditary diseases associated with DNA repair defects. (orig.) [de

The structure of DNA by direct imaging

KAUST Repository

Marini, Monica

2015-08-28

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

The structure of DNA by direct imaging

KAUST Repository

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

2015-01-01

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

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

International Nuclear Information System (INIS)

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.

2002-01-01

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)

DNA fingerprinting, biological and chemical investigation of certain Yucca species.

Science.gov (United States)

El Hawary, Seham; El Sayed, Abeer; Helmy, Maged W; El Naggar, El Moataz Bellah; Marzouk, Hanan S; Bassam, Samar M

2018-01-05

Yucca aloifolia, Y. aloifolia variegata, Y. elephantipes and Y. filamentosa were investigated. DNA sequencing was performed for the four plants and a genomic DNA fingerprint was obtained and provided. The cytotoxic activities against four human cancer cell lines were investigated. The ethanolic extracts of leaves of Y. aloifolia variegata prevailed, especially against liver cancer HepG-2 and breast cancer MCF-7. In vivo assessment of hepatoprotective activity in rats also revealed the hepatoprotective potential of the ethanolic extracts of the four plants against CCl 4 - induced rats' liver damage. Qualitative and quantitative analysis of the flavonoid and phenolic content of the promising species was performed using HPLC. The analysis identified and quantified 18 flavonoids and 19 phenolic acids in the different fractions of Y. aloifolia variegata, among which the major flavonoids were hesperidin and kaemp-3-(2-p-coumaroyl) glucose and the major phenolic acids were gallic acid and protocatechuic acid.

Quantitative phosphoproteomics to characterize signaling networks

DEFF Research Database (Denmark)

Rigbolt, Kristoffer T G; Blagoev, Blagoy

2012-01-01

for analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify......Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform...... and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments...

DNA nanotechnology from the test tube to the cell.

Science.gov (United States)

Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A; Seelig, Georg

2015-09-01

The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

DNA nanotechnology from the test tube to the cell

Science.gov (United States)

Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

2015-09-01

The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

Water versus DNA A new deal for proton transport modeling in biological matter

International Nuclear Information System (INIS)

Champion, C; Quinto, M A; Monti, J M; Galassi, M E; Fojón, O A; Hanssen, J; Rivarola, R D; Week, P F

2015-01-01

Water vapor is a common surrogate of DNA for modeling the proton-induced ionizing processes in living tissue exposed to radiations. The present study aims at scrutinizing the validity of this approximation and then revealing new insights into proton-induced energy transfers by a comparative analysis between water and realistic biological medium. In this context, self-consistent quantum mechanical modeling of the ionization and electron capture processes is reported within the continuum distorted wave-eikonal initial state framework for both isolated water molecules and DNA components impacted by proton beams. (paper)

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

Science.gov (United States)

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Modeling Cancer Metastasis using Global, Quantitative and Integrative Network Biology

DEFF Research Database (Denmark)

Schoof, Erwin; Erler, Janine

understanding of molecular processes which are fundamental to tumorigenesis. In Article 1, we propose a novel framework for how cancer mutations can be studied by taking into account their effect at the protein network level. In Article 2, we demonstrate how global, quantitative data on phosphorylation dynamics...... can be generated using MS, and how this can be modeled using a computational framework for deciphering kinase-substrate dynamics. This framework is described in depth in Article 3, and covers the design of KinomeXplorer, which allows the prediction of kinases responsible for modulating observed...... 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...

Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

Science.gov (United States)

Cox, Annie M; Goodwin, Kelly D

2013-08-15

The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

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

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...... the probability of observed peak heights and peak areas information for a pair of profiles matching the DNA mixture. Furthermore, we demonstrate how to incorporate this probability in the evaluation of the weight of the evidence by a likelihood ratio approach. Our model is based on a multivariate normal...... peak heights and areas. Complying with this latent structure, we used the EM algorithm to impute the missing variables on the basis of a compound symmetry model. The measurements were subject to intralocus and interlocus correlations not depending on the actual alleles of the DNA profiles. Owing...

Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

DEFF Research Database (Denmark)

Jensen, Pia Wrensted; Falconi, Mattia; Kristoffersen, Emil Laust

2013-01-01

structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only “signal amplification” the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1......Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very...... simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples...

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

Science.gov (United States)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

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.

Adsorption kinetics and mechanical properties of thiol-modified DNA-oligos on gold investigated by microcantilever sensors

DEFF Research Database (Denmark)

Marie, Rodolphe Charly Willy; Jensenius, Henriette; Thaysen, Jacob

2002-01-01

on the gold surface after which a significant unspecific adsorption takes place on top of the first DNA-oligo layer. The cantilever-based sensor principle has a wide range of applications in real-time local monitoring of chemical and biological interactions as well as in the detection of specific DNA......Immobilised DNA-oligo layers are scientifically and technologically appealing for a wide range of sensor applications such as DNA chips. Using microcantilever-based sensors with integrated readout, we demonstrate in situ quantitative studies of surface-stress formation during self-assembly of a 25...

Quantitative characterization of nanoparticle agglomeration within biological media

International Nuclear Information System (INIS)

Hondow, Nicole; Brydson, Rik; Wang, Peiyi; Holton, Mark D.; Brown, M. Rowan; Rees, Paul; Summers, Huw D.; Brown, Andy

2012-01-01

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.

KEY COMPARISON: CCQM-K61: Quantitation of a linearised plasmid DNA, based on a matched standard in a matrix of non-target DNA

Science.gov (United States)

Woolford, Alison; Holden, Marcia; Salit, Marc; Burns, Malcolm; Ellison, Stephen L. R.

2009-01-01

Key comparison CCQM-K61 was performed to demonstrate and document the capability of interested national metrology institutes in the determination of the quantity of specific DNA target in an aqueous solution. The study provides support for the following measurement claim: "Quantitation of a linearised plasmid DNA, based on a matched standard in a matrix of non-target DNA". The comparison was an activity of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière and was coordinated by NIST (Gaithersburg, USA) and LGC (Teddington, UK). The following laboratories (in alphabetical order) participated in this key comparison. DMSC (Thailand); IRMM (European Union); KRISS (Republic of Korea); LGC (UK); NIM (China); NIST (USA); NMIA (Australia); NMIJ (Japan); VNIIM (Russian Federation) Good agreement was observed between the reported results of all nine of the participants. Uncertainty estimates did not account fully for the dispersion of results even after allowance for possible inhomogeneity in calibration materials. Preliminary studies suggest that the effects of fluorescence threshold setting might contribute to the excess dispersion, and further study of this topic is suggested 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).

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.

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

Science.gov (United States)

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods.

Science.gov (United States)

Piñol, J; Mir, G; Gomez-Polo, P; Agustí, N

2015-07-01

The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer-template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer-template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer-template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region. © 2014 John Wiley & Sons Ltd.

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering.

Science.gov (United States)

Haddad, Yazan; Dostalova, Simona; Kudr, Jiri; Zitka, Ondrej; Heger, Zbynek; Adam, Vojtech

2017-11-09

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

Historic and current hepatitis B viral DNA and quantitative HBsAg level are not associated with cirrhosis in non-Asian women with chronic hepatitis B.

Science.gov (United States)

Harkisoen, S; Arends, J E; van den Hoek, J A R; Whelan, J; van Erpecum, K J; Boland, G J; Hoepelman, A I M

2014-12-01

Some studies done in Asian patients have shown that serum levels of hepatitis B virus (HBV) DNA predict the development of cirrhosis. However, it is unclear whether this also applies for non-Asian patients. This study investigated historic and current HBV DNA and quantitative hepatitis B surface antigen (HBsAg) levels as predictors of cirrhosis in non-Asian women with chronic HBV. A retrospective cohort study of non-Asian women with chronic HBV was performed. Among other variables, HBV DNA and quantitative HBsAg levels were measured in stored historic serum samples obtained during pregnancy (period 1990-2004) and current serum samples (period 2011-2012) to determine any association with liver cirrhosis by liver stiffness measurement (LSM). One hundred and nineteen asymptomatic, treatment-naïve non-Asian women were included; the median number of years between the historic sample and the current sample was 17 (interquartile range (IQR) 13-20). The median historic log HBV DNA and quantitative log HBsAg levels were 2.5 (IQR 1.9-3.4) IU/ml and 4.2 (IQR 3.6-4.5) IU/ml, respectively. LSM diagnosed 14 patients (12%) with F3-F4 fibrosis, i.e. stiffness >8.1kPa. No association of cirrhosis was found with historic HBV DNA (relative risk (RR) 0.34, 95% confidence interval (CI) 0.05-2.44) or with the quantitative HBsAg level (HBsAg level >1000 IU/ml, RR 0.35, 95% CI 0.11-1.11). Multivariable analysis identified alcohol consumption (odds ratio (OR) 6.4, 95% CI 1.3-30.1), aspartate aminotransferase >0.5 times the upper limit of normal (OR 15.4, 95% CI 1.9-122.6), and prothrombin time (OR 12.0, 95% CI 1.2-120.4), but not HBV DNA or quantitative HBsAg level, to be independent predictors of the presence of cirrhosis. Neither historic nor current HBV DNA or the quantitative HBsAg level is associated with the development of HBV-related cirrhosis in non-Asian women. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

Science.gov (United States)

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

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

A novel technique with enhanced detection and quantitation of HPV-16 E1- and E2-mediated DNA replication

International Nuclear Information System (INIS)

Taylor, Ewan R.; Morgan, Iain M.

2003-01-01

Transient DNA replication assays to detect papillomavirus E1/E2-mediated DNA replication have depended upon Southern blotting. This technique is hazardous (radioactive), labour intensive, semiquantitative, and physically limited in the number of samples that can be processed at any one time. We have overcome these problems by developing a real-time PCR protocol for the detection of E1/E2-mediated transient DNA replication. The results demonstrate detection of replication at levels not seen using Southern blotting demonstrating enhanced sensitivity. This technique is also, by definition, highly quantitative. Therefore, the real-time PCR technique is the optimal method for the detection of E1/E2-mediated DNA replication

Connecting qualitative observation and quantitative measurement for enhancing quantitative literacy in plant anatomy course

Science.gov (United States)

Nuraeni, E.; Rahmat, A.

2018-05-01

Forming of cognitive schemes of plant anatomy concepts is performed by processing of qualitative and quantitative data obtained from microscopic observations. To enhancing student’s quantitative literacy, strategy of plant anatomy course was modified by adding the task to analyze quantitative data produced by quantitative measurement of plant anatomy guided by material course. Participant in this study was 24 biology students and 35 biology education students. Quantitative Literacy test, complex thinking in plant anatomy test and questioner used to evaluate the course. Quantitative literacy capability data was collected by quantitative literacy test with the rubric from the Association of American Colleges and Universities, Complex thinking in plant anatomy by test according to Marzano and questioner. Quantitative literacy data are categorized according to modified Rhodes and Finley categories. The results showed that quantitative literacy of biology education students is better than biology students.

Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR.

Science.gov (United States)

Nielsen, Martin K; Peterson, David S; Monrad, Jesper; Thamsborg, Stig M; Olsen, Susanne N; Kaplan, Ray M

2008-03-01

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can

New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

Science.gov (United States)

Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

2012-01-01

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

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.

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’.

Radiation physics, biophysics, and radiation biology

International Nuclear Information System (INIS)

Hall, E.J.; Zaider, M.

1993-05-01

Research at the Center for Radiological Research is a multidisciplenary blend of physics, chemistry and biology aimed at understanding the mechanisms involved in the health problems resulting from human exposure to ionizing radiations. The focus is increased on biochemistry and the application of the techniques of molecular biology to the problems of radiation biology. Research highlights of the program from the past year are described. A mathematical model describing the production of single-strand and double-strand breaks in DNA as a function radiation quality has been completed. For the first time Monte Carlo techniques have been used to obtain directly the spatial distribution of DNA moieties altered by radiation. This information was obtained by including the transport codes a realistic description of the electronic structure of DNA. We have investigated structure activity relationships for the potential oncogenicity of a new generation of bioreductive drugs that function as hypoxic cytotoxins. Experimental and theoretical investigation of the inverse dose rate effect, whereby medium LET radiations actually produce an c effect when the dose is protracted, is now at a point where the basic mechanisms are reasonably understood and the complex interplay between dose, dose rate and radiation quality which is necessary for the effect to be present can now be predicted at least in vitro. In terms of early radiobiological damage, a quantitative link has been established between basic energy deposition and locally multiply damaged sites, the radiochemical precursor of DNA double strand breaks; specifically, the spatial and energy deposition requirements necessary to form LMDs have been evaluated. For the first time, a mechanically understood ''biological fingerprint'' of high-LET radiation has been established. Specifically measurement of the ratio of inter-to intra-chromosomal aberrations produces a unique signature from alpha-particles or neutrons

Radiation physics, biophysics, and radiation biology

Energy Technology Data Exchange (ETDEWEB)

Hall, E.J.; Zaider, M.

1993-05-01

Research at the Center for Radiological Research is a multidisciplenary blend of physics, chemistry and biology aimed at understanding the mechanisms involved in the health problems resulting from human exposure to ionizing radiations. The focus is increased on biochemistry and the application of the techniques of molecular biology to the problems of radiation biology. Research highlights of the program from the past year are described. A mathematical model describing the production of single-strand and double-strand breaks in DNA as a function radiation quality has been completed. For the first time Monte Carlo techniques have been used to obtain directly the spatial distribution of DNA moieties altered by radiation. This information was obtained by including the transport codes a realistic description of the electronic structure of DNA. We have investigated structure activity relationships for the potential oncogenicity of a new generation of bioreductive drugs that function as hypoxic cytotoxins. Experimental and theoretical investigation of the inverse dose rate effect, whereby medium LET radiations actually produce an c effect when the dose is protracted, is now at a point where the basic mechanisms are reasonably understood and the complex interplay between dose, dose rate and radiation quality which is necessary for the effect to be present can now be predicted at least in vitro. In terms of early radiobiological damage, a quantitative link has been established between basic energy deposition and locally multiply damaged sites, the radiochemical precursor of DNA double strand breaks; specifically, the spatial and energy deposition requirements necessary to form LMDs have been evaluated. For the first time, a mechanically understood biological fingerprint'' of high-LET radiation has been established. Specifically measurement of the ratio of inter-to intra-chromosomal aberrations produces a unique signature from alpha-particles or neutrons.

Automated quantitative assessment of proteins' biological function in protein knowledge bases.

Science.gov (United States)

Mayr, Gabriele; Lepperdinger, Günter; Lackner, Peter

2008-01-01

Primary protein sequence data are archived in databases together with information regarding corresponding biological functions. In this respect, UniProt/Swiss-Prot is currently the most comprehensive collection and it is routinely cross-examined when trying to unravel the biological role of hypothetical proteins. Bioscientists frequently extract single entries and further evaluate those on a subjective basis. In lieu of a standardized procedure for scoring the existing knowledge regarding individual proteins, we here report about a computer-assisted method, which we applied to score the present knowledge about any given Swiss-Prot entry. Applying this quantitative score allows the comparison of proteins with respect to their sequence yet highlights the comprehension of functional data. pfs analysis may be also applied for quality control of individual entries or for database management in order to rank entry listings.

Automated Quantitative Assessment of Proteins' Biological Function in Protein Knowledge Bases

Directory of Open Access Journals (Sweden)

Gabriele Mayr

2008-01-01

Full Text Available Primary protein sequence data are archived in databases together with information regarding corresponding biological functions. In this respect, UniProt/Swiss-Prot is currently the most comprehensive collection and it is routinely cross-examined when trying to unravel the biological role of hypothetical proteins. Bioscientists frequently extract single entries and further evaluate those on a subjective basis. In lieu of a standardized procedure for scoring the existing knowledge regarding individual proteins, we here report about a computer-assisted method, which we applied to score the present knowledge about any given Swiss-Prot entry. Applying this quantitative score allows the comparison of proteins with respect to their sequence yet highlights the comprehension of functional data. pfs analysis may be also applied for quality control of individual entries or for database management in order to rank entry listings.

Flexible automated approach for quantitative liquid handling of complex biological samples.

Science.gov (United States)

Palandra, Joe; Weller, David; Hudson, Gary; Li, Jeff; Osgood, Sarah; Hudson, Emily; Zhong, Min; Buchholz, Lisa; Cohen, Lucinda H

2007-11-01

A fully automated protein precipitation technique for biological sample preparation has been developed for the quantitation of drugs in various biological matrixes. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and controls from a Watson laboratory information management system generated work list, shaking of 96-well plates, and vacuum application. Processing time is less than 30 s per sample or approximately 45 min per 96-well plate, which is then immediately ready for injection onto an LC-MS/MS system. An overview of the process workflow is discussed, including the software development. Validation data are also provided, including specific liquid class data as well as comparative data of automated vs manual preparation using both quality controls and actual sample data. The efficiencies gained from this automated approach are described.

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

Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

Science.gov (United States)

Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

2016-05-06

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

Science.gov (United States)

Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

2004-01-01

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

DNA nanotechnology

Science.gov (United States)

Seeman, Nadrian C.; Sleiman, Hanadi F.

2018-01-01

DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Directory of Open Access Journals (Sweden)

Jie Zhu

Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

Science.gov (United States)

Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Science.gov (United States)

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Quantitative X-ray analysis of biological fluids: the microdroplet technique

International Nuclear Information System (INIS)

Roinel, N.

1988-01-01

X-ray microanalysis can be used to quantitatively determine the elemental composition of microvolumes of biological fluids. This article describes the various steps in preparation of microdroplets for analysis: The manufacturing of micropipettes, the preparation of the specimen support, the deposition of droplets on the support, shock-freezing, and lyophilization. Examples of common artifacts (incomplete rehydration prior to freezing or partial rehydration after lyophilization) are demonstrated. Analysis can be carried out either by wavelength-dispersive analysis, which is the most sensitive method, or by energy-dispersive analysis, which is more commonly available. The minimum detectable concentration is 0.05 mmol.liter-1 for 0.1-nl samples analyzed by wavelength-dispersive spectrometry and 0.5-1 mmol.liter-1 for samples analyzed by energy-dispersive spectrometry. A major problem, especially in wavelength-dispersive analysis, where high beam currents are used, is radiation damage to the specimen; in particular chloride (but also other elements) can be lost. Quantitative analysis requires the use of standard solutions with elemental concentration in the same range as those present in the specimen

Growing trend of CE at the omics level: the frontier of systems biology--an update.

Science.gov (United States)

Ban, Eunmi; Park, Soo Hyun; Kang, Min-Jung; Lee, Hyun-Jung; Song, Eun Joo; Yoo, Young Sook

2012-01-01

Omics is the study of proteins, peptides, genes, and metabolites in living organisms. Systems biology aims to understand the system through the study of the relationship between elements such as genes and proteins in biological system. Recently, systems biology emerged as the result of the advanced development of high-throughput analysis technologies such as DNA sequencers, DNA arrays, and mass spectrometry for omics studies. Among a number of analytical tools and technologies, CE and CE coupled to MS are promising and relatively rapidly developing tools with the potential to provide qualitative and quantitative analyses of biological molecules. With an emphasis on CE for systems biology, this review summarizes the method developments and applications of CE for the genomic, transcriptomic, proteomic, and metabolomic studies focusing on the drug discovery and disease diagnosis and therapies since 2009. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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

The cyclopurine deoxynucleosides: DNA repair, biological effects, mechanistic insights, and unanswered questions.

Science.gov (United States)

Brooks, Philip J

2017-06-01

Patients with the genetic disease xeroderma pigmentosum (XP) who lack the capacity to carry out nucleotides excision repair (NER) have a dramatically elevated risk of skin cancer on sun exposed areas of the body. NER is the DNA repair mechanism responsible for the removal of DNA lesions resulting from ultraviolet light. In addition, a subset of XP patients develop a progressive neurodegenerative disease, referred to as XP neurologic disease, which is thought to be the result of accumulation of endogenous DNA lesions that are repaired by NER but not other repair pathways. The 8,5-cyclopurine deoxynucleotides (cyPu) have emerged as leading candidates for such lesions, in that they result from the reaction of the hydroxyl radical with DNA, are strong blocks to transcription in human cells, and are repaired by NER but not base excision repair. Here I present a focused perspective on progress into understating the repair and biological effects of these lesions. In doing so, I emphasize the role of Tomas Lindahl and his laboratory in stimulating cyPu research. I also include a critical evaluation of the evidence supporting a role for cyPu lesions in XP neurologic disease, with a focus on outstanding questions, and conceptual and technologic challenges. Copyright © 2017. Published by Elsevier Inc.

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.

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

Science.gov (United States)

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

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. © 2014 Elsevier Inc. All rights reserved.

Integrating plant and animal biology for the search of novel DNA damage biomarkers

Czech Academy of Sciences Publication Activity Database

Nikitaki, Z.; Holá, Marcela; Donà, M.; Pavlopoulou, A.; Michalopoulos, I.; Angelis, Karel; Georgakilas, A. G.; Macovei, I.; Balestrazzi, A.

2018-01-01

Roč. 775, JAN-MAR (2018), s. 21-38 ISSN 1383-5742 R&D Projects: GA ČR GA16-01137S Institutional support: RVO:61389030 Keywords : DNA damage response * Ionizing radiation * Radiation exposure monitoring * Radiotolerance * Ultraviolet radiation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Genetics and heredity (medical genetics to be 3) Impact factor: 5.500, year: 2016

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.

The nucleic acid revolution continues - will forensic biology become forensic molecular biology?

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Gunn, Peter; Walsh, Simon; Roux, Claude

2014-01-01

Molecular biology has evolved far beyond that which could have been predicted at the time DNA identity testing was established. Indeed we should now perhaps be referring to "forensic molecular biology." Aside from DNA's established role in identifying the "who" in crime investigations, other developments in medical and developmental molecular biology are now ripe for application to forensic challenges. The impact of DNA methylation and other post-fertilization DNA modifications, plus the emerging role of small RNAs in the control of gene expression, is re-writing our understanding of human biology. It is apparent that these emerging technologies will expand forensic molecular biology to allow for inferences about "when" a crime took place and "what" took place. However, just as the introduction of DNA identity testing engendered many challenges, so the expansion of molecular biology into these domains will raise again the issues of scientific validity, interpretation, probative value, and infringement of personal liberties. This Commentary ponders some of these emerging issues, and presents some ideas on how they will affect the conduct of forensic molecular biology in the foreseeable future.

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

Directory of Open Access Journals (Sweden)

Sabrina Schreiner

2017-05-01

Full Text Available Chronic hepatitis B virus (HBV infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR, a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.

Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings

International Nuclear Information System (INIS)

Quaite, F.E.; Sutherland, B.M.; Sutherland, J.C.

1991-01-01

Depletion of stratospheric ozone will increase the solar ultraviolet radiation in the range from 290-320 nm (UVB) that reaches the surface of the earth, placing an increased UV burden on exposed organisms. One consequence of increased UVB may be decreased productivity of crop plants. A principal lesion caused by UV in DNA is the cyclobutyl pyrimidine dimer. We have adapted a method for measuring these dimers in nanogram quantities of non-radioactive DNA for use in UV-irradiated plants. We find that biologically relevant doses of broad band UVB radiation induce easily detectable frequencies of pyrimidine dimers in the DNA of irradiated alfalfa sprout leaves and that the dose response for dimer formation is linear up to doses of at least 690 J/m 2 . We also find easily measurable frequencies of dimers in the leaves of seedlings grown in glass filtered sunlight but not exposed to additional UVB, suggesting that significant number of dimers are formed in plants exposed to normal sunlight. 27 refs., 3 figs., 1 tab

Quantitative HPLC determination of [99mTc]-pertechnetate in radiopharmaceuticals and biological samples: Pt. 1

International Nuclear Information System (INIS)

Tianze Zhou; Hirth, W.W.; Heineman, W.R.; Deutsch, Edward

1988-01-01

Techniques have been developed which allow HPLC (high performance liquid chromatography) to be used for the quantitative determination of [ 99m Tc]pertechnetate in radiopharmaceuticals and biological samples. An instrumental technique accounts for 99m Tc species which do not elute from the HPLC column, while a chemical technique obviates interferences caused by Sn(II). These two techniques are incorporated into an anion exchange HPLC procedure which is applied to the determination of [ 99m Tc]pertechnetate in 99m Tc-diphosphonate radiopharmaceuticals and biological samples. (author)

The impact of a scheduling change on ninth grade high school performance on biology benchmark exams and the California Standards Test

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Leonardi, Marcelo

The primary purpose of this study was to examine the impact of a scheduling change from a trimester 4x4 block schedule to a modified hybrid schedule on student achievement in ninth grade biology courses. This study examined the impact of the scheduling change on student achievement through teacher created benchmark assessments in Genetics, DNA, and Evolution and on the California Standardized Test in Biology. The secondary purpose of this study examined the ninth grade biology teacher perceptions of ninth grade biology student achievement. Using a mixed methods research approach, data was collected both quantitatively and qualitatively as aligned to research questions. Quantitative methods included gathering data from departmental benchmark exams and California Standardized Test in Biology and conducting multiple analysis of covariance and analysis of covariance to determine significance differences. Qualitative methods include journal entries questions and focus group interviews. The results revealed a statistically significant increase in scores on both the DNA and Evolution benchmark exams. DNA and Evolution benchmark exams showed significant improvements from a change in scheduling format. The scheduling change was responsible for 1.5% of the increase in DNA benchmark scores and 2% of the increase in Evolution benchmark scores. The results revealed a statistically significant decrease in scores on the Genetics Benchmark exam as a result of the scheduling change. The scheduling change was responsible for 1% of the decrease in Genetics benchmark scores. The results also revealed a statistically significant increase in scores on the CST Biology exam. The scheduling change was responsible for .7% of the increase in CST Biology scores. Results of the focus group discussions indicated that all teachers preferred the modified hybrid schedule over the trimester schedule and that it improved student achievement.

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

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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.

Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

DEFF Research Database (Denmark)

Stangegaard, Michael; Jørgensen, Mads; Hansen, Anders Johannes

2009-01-01

, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpFlSTR« Identifiler« and AmpFlSTR« Yfiler« (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types......We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs...... of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025...

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

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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.

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

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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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

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Yankson, Kweku K.; Steck, Todd R.

2009-01-01

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

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.

DNA origami-based standards for quantitative fluorescence microscopy.

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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.

An information gap in DNA evidence interpretation.

Directory of Open Access Journals (Sweden)

Mark W Perlin

Full Text Available Forensic DNA evidence often contains mixtures of multiple contributors, or is present in low template amounts. The resulting data signals may appear to be relatively uninformative when interpreted using qualitative inclusion-based methods. However, these same data can yield greater identification information when interpreted by computer using quantitative data-modeling methods. This study applies both qualitative and quantitative interpretation methods to a well-characterized DNA mixture and dilution data set, and compares the inferred match information. The results show that qualitative interpretation loses identification power at low culprit DNA quantities (below 100 pg, but that quantitative methods produce useful information down into the 10 pg range. Thus there is a ten-fold information gap that separates the qualitative and quantitative DNA mixture interpretation approaches. With low quantities of culprit DNA (10 pg to 100 pg, computer-based quantitative interpretation provides greater match sensitivity.

Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice.

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Antoch, Marina P; Wrobel, Michelle; Kuropatwinski, Karen K; Gitlin, Ilya; Leonova, Katerina I; Toshkov, Ilia; Gleiberman, Anatoli S; Hutson, Alan D; Chernova, Olga B; Gudkov, Andrei V

2017-03-19

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

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Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

2013-04-10

Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

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

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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.

Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection.

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Caviglia, Gian Paolo; Abate, Maria Lorena; Tandoi, Francesco; Ciancio, Alessia; Amoroso, Antonio; Salizzoni, Mauro; Saracco, Giorgio Maria; Rizzetto, Mario; Romagnoli, Renato; Smedile, Antonina

2018-04-02

The accurate diagnosis of occult HBV infection (OBI) requires the demonstration of HBV DNA in liver biopsies of HBsAg-negative subjects. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the HB-core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive subjects. The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R 2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/10 5 cells, respectively). A true OBI status was found in 52% (52/100) of the subjects and cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/10 5 cells (95% confidence interval 5-25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (5.7 [3.6-9.7] vs. 17.0 [7.0-39.2] COI, p = 0.007). By multivariate analysis, an anti-HBc IgG value above a 4.4 cut-off index (COI) was associated with the finding of intrahepatic HBV cccDNA (OR = 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%. With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients. The covalently closed circular DNA (cccDNA) form of the Hepatitis B virus (HBV) sustains the persistence of the virus even after decades of resolution of the florid infection (Occult HBV infection=OBI). In the present study we developed an highly sensitive method based on droplet digital PCR technology for the detection

Historic and current hepatitis B viral DNA and quantitative HBsAg level are not associated with cirrhosis in non-Asian women with chronic hepatitis B

NARCIS (Netherlands)

Harkisoen, S.; Arends, J. E.; van den Hoek, J. A. R.; Whelan, J.; van Erpecum, K. J.; Boland, G. J.; Hoepelman, A. I. M.

2014-01-01

Some studies done in Asian patients have shown that serum levels of hepatitis B virus (HBV) DNA predict the development of cirrhosis. However, it is unclear whether this also applies for non-Asian patients. This study investigated historic and current HBV DNA and quantitative hepatitis B surface

Quantitative PCR is a Valuable Tool to Monitor the Performance of DNA-Encoded Chemical Library Selections.

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Li, Yizhou; Zimmermann, Gunther; Scheuermann, Jörg; Neri, Dario

2017-05-04

Phage-display libraries and DNA-encoded chemical libraries (DECLs) represent useful tools for the isolation of specific binding molecules from large combinatorial sets of compounds. With both methods, specific binders are recovered at the end of affinity capture procedures by using target proteins of interest immobilized on a solid support. However, although the efficiency of phage-display selections is routinely quantified by counting the phage titer before and after the affinity capture step, no similar quantification procedures have been reported for the characterization of DECL selections. In this article, we describe the potential and limitations of quantitative PCR (qPCR) methods for the evaluation of selection efficiency by using a combinatorial chemical library with more than 35 million compounds. In the experimental conditions chosen for the selections, a quantification of DNA input/recovery over five orders of magnitude could be performed, revealing a successful enrichment of abundant binders, which could be confirmed by DNA sequencing. qPCR provided rapid information about the performance of selections, thus facilitating the optimization of experimental conditions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seroprevalence, molecular epidemiology and quantitation of parvovirus B19 DNA levels in Iranian blood donors.

Science.gov (United States)

Zadsar, Maryam; Aghakhani, Arezoo; Banifazl, Mohammad; Kazemimanesh, Monireh; Tabatabaei Yazdi, Seyed Morteza; Mamishi, Setareh; Bavand, Anahita; Sadat Larijani, Mona; Ramezani, Amitis

2018-04-16

Human parvovirus B19 (B19) infection is common among blood donors, and healthy blood donors can transmit virus via transfusion. Due to resistance of B19 to viral inactivation methods, there is a potential concern regarding transfusion safety in blood products. We aimed to determine the seroprevalence, molecular epidemiology, and quantitation of B19 DNA levels in blood donors in Tehran, Iran. A total of 500 blood donors from Blood Transfusion Research Center were studied. ELISA was used for detection of B19 IgG and IgM and nested PCR was carried out for detection of B19 DNA. PCR products were subjected to direct sequencing. B19 viral load was determined by real time PCR. B19 IgG, IgM, and DNA were detected in 27.6, 2.6, and 1.2% of donors respectively. Ten samples (2%) were positive for both antibodies while in four cases (0.8%), B19 IgG and DNA detected simultaneously. One case had B19 IgM, IgG, and viremia concurrently. The titers of B19 DNA in four of six donors were more than 10 6  IU/mL (high level viremia) and all four cases had IgG simultaneously. All B19 isolates categorized in genotype 1A. Our findings indicated that prevalence of B19 DNA in Iranian blood donors was comparable with previous studies throughout the world. High level B19 viremia found in 0.8% of our donors and all viremic donors revealed neutralizing B19 antibody. Therefore implementation of a B19 screening test for each volunteer blood donor does not appear to be necessary but B19 testing for plasma-derived products seems important in Iranian donors. © 2018 Wiley Periodicals, Inc.

Application of LC–MS/MS for quantitative analysis of glucocorticoids and stimulants in biological fluids

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Jamshed Haneef

2013-10-01

Full Text Available 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. Keywords: LC–MS/MS, Ionization techniques, Glucocorticoids, Stimulants, Hyphenated techniques, Biological fluid

The ultraviolet environment of Mars: biological implications past, present, and future.

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Cockell, C S; Catling, D C; Davis, W L; Snook, K; Kepner, R L; Lee, P; McKay, C P

2000-08-01

A radiative transfer model is used to quantitatively investigate aspects of the martian ultraviolet radiation environment, past and present. Biological action spectra for DNA inactivation and chloroplast (photosystem) inhibition are used to estimate biologically effective irradiances for the martian surface under cloudless skies. Over time Mars has probably experienced an increasingly inhospitable photobiological environment, with present instantaneous DNA weighted irradiances 3.5-fold higher than they may have been on early Mars. This is in contrast to the surface of Earth, which experienced an ozone amelioration of the photobiological environment during the Proterozoic and now has DNA weighted irradiances almost three orders of magnitude lower than early Earth. Although the present-day martian UV flux is similar to that of early Earth and thus may not be a critical limitation to life in the evolutionary context, it is a constraint to an unadapted biota and will rapidly kill spacecraft-borne microbes not covered by a martian dust layer. Microbial strategies for protection against UV radiation are considered in the light of martian photobiological calculations, past and present. Data are also presented for the effects of hypothetical planetary atmospheric manipulations on the martian UV radiation environment with estimates of the biological consequences of such manipulations.

The ultraviolet environment of Mars: biological implications past, present, and future

Science.gov (United States)

Cockell, C. S.; Catling, D. C.; Davis, W. L.; Snook, K.; Kepner, R. L.; Lee, P.; McKay, C. P.

2000-01-01

A radiative transfer model is used to quantitatively investigate aspects of the martian ultraviolet radiation environment, past and present. Biological action spectra for DNA inactivation and chloroplast (photosystem) inhibition are used to estimate biologically effective irradiances for the martian surface under cloudless skies. Over time Mars has probably experienced an increasingly inhospitable photobiological environment, with present instantaneous DNA weighted irradiances 3.5-fold higher than they may have been on early Mars. This is in contrast to the surface of Earth, which experienced an ozone amelioration of the photobiological environment during the Proterozoic and now has DNA weighted irradiances almost three orders of magnitude lower than early Earth. Although the present-day martian UV flux is similar to that of early Earth and thus may not be a critical limitation to life in the evolutionary context, it is a constraint to an unadapted biota and will rapidly kill spacecraft-borne microbes not covered by a martian dust layer. Microbial strategies for protection against UV radiation are considered in the light of martian photobiological calculations, past and present. Data are also presented for the effects of hypothetical planetary atmospheric manipulations on the martian UV radiation environment with estimates of the biological consequences of such manipulations.

DNA to DNA transcription might exist in eukaryotic cells

OpenAIRE

Li, Gao-De

2016-01-01

Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

Biological defense mechanisms against DNA double-strand break and their possible medical applications

International Nuclear Information System (INIS)

Matsumoto, Yoshihisa

2011-01-01

Radiation is now widely used for clinical diagnosis and therapeutics. On the other hand, radiation influences various tissues represented by immunological and reproductive systems, and is also recognized as one of the cause of carcinogenesis. Such pleiotropic effects of radiation are mediated through generation of damages on DNA molecule, vitally important genetic macromolecule. Among various types of DNA damages, double-strand break (DSB) is considered most critical and, therefore, responsible for biological effects. DSB is repaired mainly through two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Understanding of these mechanisms has been greatly deepened in past 20 years and is now providing a promising approach toward cancer therapy. We have studied the mechanisms of NHEJ, focusing especially on the role of phosphorylation and the assembly of machinery therein, which will be introduced below. (author)

Human Chromosome 7: DNA Sequence and Biology

OpenAIRE

Scherer, Stephen W.; Cheung, Joseph; MacDonald, Jeffrey R.; Osborne, Lucy R.; Nakabayashi, Kazuhiko; Herbrick, Jo-Anne; Carson, Andrew R.; Parker-Katiraee, Layla; Skaug, Jennifer; Khaja, Razi; Zhang, Junjun; Hudek, Alexander K.; Li, Martin; Haddad, May; Duggan, Gavin E.

2003-01-01

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate gene...

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

Science.gov (United States)

Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

2007-01-01

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can

Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

Science.gov (United States)

Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

2000-01-01

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

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.

Role of excision repair in postradiation recovery of biological activity of cellular DNA Bacillus subtilis

International Nuclear Information System (INIS)

Filippov, V.D.

1976-01-01

DNA extracted from UV-irradiated prototroph cells of Bacillus subtilis uvr + (45 sec. of UV light, 20% survivals) has a lowered transforming activity (TA) of markers purB and metB, and a lowered ratio TA pur/TA met. During the subsequent incubation of uvr + cells in glucose-salt medium free of nitrogen sources the TA of markers and the ratio between them increase. No increase is observed during the postradiation incubation under the same conditions or in a nutrition medium of uvr cells, deficient in escision of pyrimidine dimers. The increment of DNA begins approsimately in 30 min. after the beginning of incubation of irradiated uvr cells in nutrition medium. On the basis of these facts it is concluded that neither the replication of damaged DNA nor the postreplication repair, but only excision repair, can provide the recovery of biological (transforming) activity of cellular DNA in Bac. subtilis. The system given might be a suitable model for testing compounds which affect the activity of this process. The well-known inhibitors of dark repair, caffeine, proflavine to inhibit reversibly the initial steps of the process/ and especially acriflavine, delay the recovery of markers of cellular DNA in irradiated uvr + cells. Caffeine is proved to inhibit reversibly the initial steps of the process

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

Science.gov (United States)

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

International Nuclear Information System (INIS)

Klajic, Jovana; Tost, Jörg; Kristensen, Vessela N; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise

2013-01-01

Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

Science.gov (United States)

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Liquid chromatography coupled with tandem mass spectrometry for the quantitative analysis of anticancer drugs in biological matrices

NARCIS (Netherlands)

Stokvis, Ellen

2004-01-01

In this thesis, the development and validation of liquid chromatography tandem mass spectrometric (LC-MS/MS) methods for the quantitative bioanalysis of anticancer drugs are described. The monitoring of these drugs in biological fluids and tissues is important during both pre-clinical and clinical

Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

Science.gov (United States)

Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

2014-05-01

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

Science.gov (United States)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

Using environmental DNA to census marine fishes in a large mesocosm.

Directory of Open Access Journals (Sweden)

Ryan P Kelly

Full Text Available The ocean is a soup of its resident species' genetic material, cast off in the forms of metabolic waste, shed skin cells, or damaged tissue. Sampling this environmental DNA (eDNA is a potentially powerful means of assessing whole biological communities, a significant advance over the manual methods of environmental sampling that have historically dominated marine ecology and related fields. Here, we estimate the vertebrate fauna in a 4.5-million-liter mesocosm aquarium tank at the Monterey Bay Aquarium of known species composition by sequencing the eDNA from its constituent seawater. We find that it is generally possible to detect mitochondrial DNA of bony fishes sufficient to identify organisms to taxonomic family- or genus-level using a 106 bp fragment of the 12S ribosomal gene. Within bony fishes, we observe a low false-negative detection rate, although we did not detect the cartilaginous fishes or sea turtles present with this fragment. We find that the rank abundance of recovered eDNA sequences correlates with the abundance of corresponding species' biomass in the mesocosm, but the data in hand do not allow us to develop a quantitative relationship between biomass and eDNA abundance. Finally, we find a low false-positive rate for detection of exogenous eDNA, and we were able to diagnose non-native species' tissue in the food used to maintain the mesocosm, underscoring the sensitivity of eDNA as a technique for community-level ecological surveys. We conclude that eDNA has substantial potential to become a core tool for environmental monitoring, but that a variety of challenges remain before reliable quantitative assessments of ecological communities in the field become possible.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

Science.gov (United States)

Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

2014-07-01

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit

NARCIS (Netherlands)

Peters, Remco P. H.; van Agtmael, Michiel A.; Gierveld, Sonja; Danner, Sven A.; Groeneveld, A. B. Johan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

2007-01-01

Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus

Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

Science.gov (United States)

Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

2016-03-01

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

Application of LC–MS/MS for quantitative analysis of glucocorticoids and stimulants in biological fluids

OpenAIRE

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

2013-01-01

Liquid chromatography tandem mass chromatography (LCâMS/MS) is an important hyphenated technique for quantitative analysis of drugs in biological fluids. Because of high sensitivity and selectivity, LCâMS/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 acquisiti...

Application of LC–MS/MS for quantitative analysis of glucocorticoids and stimulants in biological fluids

OpenAIRE

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

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 acquisiti...

Highly functionalized piperidines: Free radical scavenging, anticancer activity, DNA interaction and correlation with biological activity

OpenAIRE

Suvankar Das; Cristiane J. da Silva; Marina de M. Silva; Maria Dayanne de A. Dantas; Ângelo de Fátima; Ana Lúcia T. Góis Ruiz; Cleiton M. da Silva; João Ernesto de Carvalho; Josué C.C. Santos; Isis M. Figueiredo; Edeildo F. da Silva-Júnior; Thiago M. de Aquino; João X. de Araújo-Júnior; Goutam Brahmachari; Luzia Valentina Modolo

2018-01-01

Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the anion radical superoxide (·O2−). The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to ·O2− scavenger was piperidine...

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.

Mammalian spermatogenesis as a biologic dosimeter for radiation

Energy Technology Data Exchange (ETDEWEB)

Hacker, U; Schumann, J; Goehde, W; Mueller, K [Muenster Univ. (Germany, F.R.)

1981-01-01

Mouse spermatogenesis was used as an in vivo test system obviously suitable as a biologic dosimeter. Ionizing irradiation induced changes in the freqency distribution of the cellular DNA content of whole testis preparations. These changes can be analysed by flow cytometry. Such measurements deliver information on: (1) an increase of the coefficient of variation of the DNA histograms of some spermatogenic cells, (2) induction of diploid mature sperm, and (3) the dose-depending inactivation of the highly sensitive differentiated spermatogonia. In this model especially No. 3 delivers quantitative information on the cytotoxic action of ionizing irradiation and chemical noxae as well, whereas Nos 1 and 2 may be used as qualitative criteria of mutagenic action of physical and chemical agents, No. 2 obviously being more sensitive than No. 1.

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 t...

Quantitative modeling of gene networks of biological systems using fuzzy Petri nets and fuzzy sets

Directory of Open Access Journals (Sweden)

Raed I. Hamed

2018-01-01

Full Text Available Quantitative demonstrating of organic frameworks has turned into an essential computational methodology in the configuration of novel and investigation of existing natural frameworks. Be that as it may, active information that portrays the framework's elements should be known keeping in mind the end goal to get pertinent results with the routine displaying strategies. This information is frequently robust or even difficult to get. Here, we exhibit a model of quantitative fuzzy rational demonstrating approach that can adapt to obscure motor information and hence deliver applicable results despite the fact that dynamic information is fragmented or just dubiously characterized. Besides, the methodology can be utilized as a part of the blend with the current cutting edge quantitative demonstrating strategies just in specific parts of the framework, i.e., where the data are absent. The contextual analysis of the methodology suggested in this paper is performed on the model of nine-quality genes. We propose a kind of FPN model in light of fuzzy sets to manage the quantitative modeling of biological systems. The tests of our model appear that the model is practical and entirely powerful for information impersonation and thinking of fuzzy expert frameworks.

Recent advances in yeast molecular biology: recombinant DNA

International Nuclear Information System (INIS)

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

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.

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.

Radiation biology as a basis for multidisciplinary cancer therapy

International Nuclear Information System (INIS)

Hosoya, N.

2017-01-01

The research field of radiation biology has progressed greatly thanks to the advances in molecular biology. DNA in the cell nucleus is the principal target of radiation. The biological effect of radiation can be determined by how the DNA damage is processed in the cell. In order to prevent deleterious biological effects due to DNA damage, the cells possess a system termed 'DNA damage response'. The DNA damage response finally induces cell cycle arrest, activation of DNA repair pathways, or cell death. If accurately repaired, DNA damage will result in survival of cells with no biological effects. If inaccurately repaired, DNA damage may result in survival of cells exhibiting genetic alterations, which can lead to the development of various diseases including cancer. If unrepaired, fatal DNA damage such as the DNA double-strand break will result in cell depth. Since radiation therapy and chemotherapy are designed to specifically kill cancer cells by inducing DNA double-strand breaks, it is important to take advantage of cancer-specific abnormalities in DNA damage response. In this review, I describe the impact of targeting DNA damage response in cancer therapy and show how progress in radiation biology has contributed to the development of novel therapeutic strategies. (author)

Neutron Reflectometry Investigations of the Interaction of DNA-PAMAM Dendrimers with Model Biological Membranes

International Nuclear Information System (INIS)

Ainalem, M.L.; Rennie, A.R.; Campbell, Richard; Edler, Karen; Nylander, Tommy

2009-01-01

The systemic delivery of DNA for gene therapy requires control of DNA compaction by an agent, such a lipid, surfactant or a polymer (e.g. cationic dendrimers) as well as understanding of how this complex interacts with a biological membrane. Poly (amido amine) (PAMAM) dendrimers have been reported to be a promising synthetic gene-transfection agent. We have studied the structure of the complexes formed between DNA and PAMAM dendrimers with SANS, dynamic light scattering and cryo-TEM. Here we noted that the structure of the complex formed strongly depends on the generation of the dendrimer. The results of the adsorption of generation 2 (G2) and 4 (G4) PAMAM dendrimers to surface deposited bilayers, consisting of palmitoyl oleoyl phosphatidyl choline on silicon surface, have been studied using neutron reflectometry (NR). The NR data shows that the dendrimers are able to penetrate the bilayer. However, the complex is less able to penetrate the bilayer, but rather stays on the top of the bilayer. The dendrimers appear slightly flattened on the surface in comparison with their size in bulk as determined by light scattering. We will also report on the interfacial behavior of the DNA-PAMAM complexes at other types of studies of interfaces, important for biomedical applications, where NR has allowed us to determine the layer structure and composition. (author)

Surprising conformers of the biologically important A·T DNA base pairs: QM/QTAIM proofs

Science.gov (United States)

Brovarets', Ol'ha O.; Tsiupa, Kostiantyn S.; Hovorun, Dmytro M.

2018-02-01

For the first time novel high-energy conformers – A·T(wWC) (5.36), A·T(wrWC) (5.97), A·T(wH) (5.78) and A·T(wrH) (ΔG=5.82 kcal•mol-1) were revealed for each of the four biologically important A·T(WC) DNA base pairs – Watson-Crick A·T(WC), reverse Watson-Crick A·T(rWC), Hoogsteen A·T(H) and reverse Hoogsteen A·T(rH) at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of quantum-mechanical theory in the continuum with ɛ=4 under normal conditions. Each of these conformers possesses substantially non-planar wobble (w) structure and is stabilized by the participation of the two anti-parallel N6H/N6H'…O4/O2 and N3H…N6 H-bonds, involving the pyramidalized amino group of the A DNA base as an acceptor and a donor of the H-bonding. The transition states – TSA·T(WC)↔A·T(wWC), TSA·T(rWC)↔A·T(wrWC), TSA·T(H)↔A·T(wH) and TSA·T(rH)↔A·T(wrH), controlling the dipole-active transformations of the conformers from the main plane-symmetric state into the high-energy, significantly non-planar state and vice versa, were localized. They also possess wobble structures similarly to the high-energy conformers and are stabilized by the participation of the N6H/N6H'…O4/O2 and N3H…N6 H-bonds. Discovered conformers of the A·T DNA base pairs are dynamically stable short-lived structures (lifetime τ = (1.4-3.9) ps). Their possible biological significance and future perspectives have been briefly discussed.

Surprising Conformers of the Biologically Important A·T DNA Base Pairs: QM/QTAIM Proofs

Directory of Open Access Journals (Sweden)

Ol'ha O. Brovarets'

2018-02-01

Full Text Available For the first time novel high-energy conformers–A·T(wWC (5.36, A·T(wrWC (5.97, A·T(wH (5.78, and A·T(wrH (ΔG = 5.82 kcal·mol−1 (See Graphical Abstract were revealed for each of the four biologically important A·T DNA base pairs – Watson-Crick A·T(WC, reverse Watson-Crick A·T(rWC, Hoogsteen A·T(H and reverse Hoogsteen A·T(rH at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p level of quantum-mechanical theory in the continuum with ε = 4 under normal conditions. Each of these conformers possesses substantially non-planar wobble (w structure and is stabilized by the participation of the two anti-parallel N6H/N6H′…O4/O2 and N3H…N6 H-bonds, involving the pyramidalized amino group of the A DNA base as an acceptor and a donor of the H-bonding. The transition states – TSA·T(WC↔A·T(wWC, TSA·T(rWC↔A·T(wrWC, TSA·T(H↔A·T(wH, and TSA·T(rH↔A·T(wrH, controlling the dipole-active transformations of the conformers from the main plane-symmetric state into the high-energy, significantly non-planar state and vice versa, were localized. They also possess wobble structures similarly to the high-energy conformers and are stabilized by the participation of the N6H/N6H′…O4/O2 and N3H…N6 H-bonds. Discovered conformers of the A·T DNA base pairs are dynamically stable short-lived structures [lifetime τ = (1.4–3.9 ps]. Their possible biological significance and future perspectives have been briefly discussed.

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.

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.

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

International Nuclear Information System (INIS)

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

2011-01-01

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 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 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.

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories

Directory of Open Access Journals (Sweden)

Rainy Jeffrey

2007-09-01

Full Text Available Abstract Background Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies. Results We have generated a program that allows complete in silico simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers. Conclusion PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction.

Synthetic Biology: Putting Synthesis into Biology

Science.gov (United States)

Liang, Jing; Luo, Yunzi; Zhao, Huimin

2010-01-01

The ability to manipulate living organisms is at the heart of a range of emerging technologies that serve to address important and current problems in environment, energy, and health. However, with all its complexity and interconnectivity, biology has for many years been recalcitrant to engineering manipulations. The recent advances in synthesis, analysis, and modeling methods have finally provided the tools necessary to manipulate living systems in meaningful ways, and have led to the coining of a field named synthetic biology. The scope of synthetic biology is as complicated as life itself – encompassing many branches of science, and across many scales of application. New DNA synthesis and assembly techniques have made routine the customization of very large DNA molecules. This in turn has allowed the incorporation of multiple genes and pathways. By coupling these with techniques that allow for the modeling and design of protein functions, scientists have now gained the tools to create completely novel biological machineries. Even the ultimate biological machinery – a self-replicating organism – is being pursued at this moment. It is the purpose of this review to dissect and organize these various components of synthetic biology into a coherent picture. PMID:21064036

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

International Nuclear Information System (INIS)

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

2014-01-01

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

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.

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.

Modeling DNA affinity landscape through two-round support vector regression with weighted degree kernels

KAUST Repository

Wang, Xiaolei

2014-12-12

Background: A quantitative understanding of interactions between transcription factors (TFs) and their DNA binding sites is key to the rational design of gene regulatory networks. Recent advances in high-throughput technologies have enabled high-resolution measurements of protein-DNA binding affinity. Importantly, such experiments revealed the complex nature of TF-DNA interactions, whereby the effects of nucleotide changes on the binding affinity were observed to be context dependent. A systematic method to give high-quality estimates of such complex affinity landscapes is, thus, essential to the control of gene expression and the advance of synthetic biology. Results: Here, we propose a two-round prediction method that is based on support vector regression (SVR) with weighted degree (WD) kernels. In the first round, a WD kernel with shifts and mismatches is used with SVR to detect the importance of subsequences with different lengths at different positions. The subsequences identified as important in the first round are then fed into a second WD kernel to fit the experimentally measured affinities. To our knowledge, this is the first attempt to increase the accuracy of the affinity prediction by applying two rounds of string kernels and by identifying a small number of crucial k-mers. The proposed method was tested by predicting the binding affinity landscape of Gcn4p in Saccharomyces cerevisiae using datasets from HiTS-FLIP. Our method explicitly identified important subsequences and showed significant performance improvements when compared with other state-of-the-art methods. Based on the identified important subsequences, we discovered two surprisingly stable 10-mers and one sensitive 10-mer which were not reported before. Further test on four other TFs in S. cerevisiae demonstrated the generality of our method. Conclusion: We proposed in this paper a two-round method to quantitatively model the DNA binding affinity landscape. Since the ability to modify

Ordinary differential equations with applications in molecular biology.

Science.gov (United States)

Ilea, M; Turnea, M; Rotariu, M

2012-01-01

Differential equations are of basic importance in molecular biology mathematics because many biological laws and relations appear mathematically in the form of a differential equation. In this article we presented some applications of mathematical models represented by ordinary differential equations in molecular biology. The vast majority of quantitative models in cell and molecular biology are formulated in terms of ordinary differential equations for the time evolution of concentrations of molecular species. Assuming that the diffusion in the cell is high enough to make the spatial distribution of molecules homogenous, these equations describe systems with many participating molecules of each kind. We propose an original mathematical model with small parameter for biological phospholipid pathway. All the equations system includes small parameter epsilon. The smallness of epsilon is relative to the size of the solution domain. If we reduce the size of the solution region the same small epsilon will result in a different condition number. It is clear that the solution for a smaller region is less difficult. We introduce the mathematical technique known as boundary function method for singular perturbation system. In this system, the small parameter is an asymptotic variable, different from the independent variable. In general, the solutions of such equations exhibit multiscale phenomena. Singularly perturbed problems form a special class of problems containing a small parameter which may tend to zero. Many molecular biology processes can be quantitatively characterized by ordinary differential equations. Mathematical cell biology is a very active and fast growing interdisciplinary area in which mathematical concepts, techniques, and models are applied to a variety of problems in developmental medicine and bioengineering. Among the different modeling approaches, ordinary differential equations (ODE) are particularly important and have led to significant advances

Conformation-dependent DNA attraction

Science.gov (United States)

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-05-01

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

Computer Simulation and Data Analysis in Molecular Biology and Biophysics An Introduction Using R

CERN Document Server

Bloomfield, Victor

2009-01-01

This book provides an introduction, suitable for advanced undergraduates and beginning graduate students, to two important aspects of molecular biology and biophysics: computer simulation and data analysis. It introduces tools to enable readers to learn and use fundamental methods for constructing quantitative models of biological mechanisms, both deterministic and with some elements of randomness, including complex reaction equilibria and kinetics, population models, and regulation of metabolism and development; to understand how concepts of probability can help in explaining important features of DNA sequences; and to apply a useful set of statistical methods to analysis of experimental data from spectroscopic, genomic, and proteomic sources. These quantitative tools are implemented using the free, open source software program R. R provides an excellent environment for general numerical and statistical computing and graphics, with capabilities similar to Matlab®. Since R is increasingly used in bioinformat...

DNA modification by alkylating compounds

Energy Technology Data Exchange (ETDEWEB)

Kruglyakova, E.E.

1985-09-01

Results are given for research on the physico-chemical properties of alkylating compounds - nitroso alkyl ureas (NAU) which possess a broad spectrum of biological activity, such as mutagenic, carcinogenic, and anti-tumor action that is due to the alkylation and carbamoylation of DNA as well as other cellular components. Identified chemical products of NAU interaction with DNA and its components are cited. Structural conversions of a DNA macromolecule resulting from its chemical modification are examined. NAU are used to discuss possible biological consequences of DNA modification. 148 references.

A Suitable Method for DNA Extraction from Bones for Forensic Applications: A Case Study

Directory of Open Access Journals (Sweden)

Aqeela S. Abuidrees

2016-06-01

Full Text Available Human identification techniques are constantly developing. Before the discovery of DNA, anthropology accompanied with odontology was the most applicable technique for human identification. With the new era of molecular biology and the revolution of DNA and PCR techniques, DNA profiling has become the core of the human forensic identification process. Different types of samples can be exploited in forensic DNA analysis. In some extreme cases, bone samples are the only accessible samples of DNA due to the bad conditions of putrefaction or degradation of other biological materials and tissues. Therefore, an appropriate method should be determined to yield a full and clean profile. A case study is presented here in order to identify human remains and conclude the most appropriate method of DNA extraction from human remains. In addition, this study looks at the best part of the skeletal remains to be considered in the extraction of DNA for the purposes of identification. A suspect admitted that he buried his aborted son six months ago. The remains were recovered and DNA analysis was performed in order to determine any genetic link of the remains to the suspect and the female who delivered the baby. Two extraction methods were compared, the standard organic (phenol:chloroform:isoamyl alcohol and automated extraction using magnetic beads coated with silica (Qiagen EZ1 Advanced XL. Two bone parts, femur and clavicle, were also compared in terms of DNA yield. The efficiency of the two methods of DNA extraction from bones is illustrated quantitatively and qualitatively. Paternity testing was performed and the suspect was excluded from being the alleged father.

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

Science.gov (United States)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Topology in Molecular Biology

CERN Document Server

Monastyrsky, Michail Ilych

2007-01-01

The book presents a class of new results in molecular biology for which topological methods and ideas are important. These include: the large-scale conformation properties of DNA; computational methods (Monte Carlo) allowing the simulation of large-scale properties of DNA; the tangle model of DNA recombination and other applications of Knot theory; dynamics of supercoiled DNA and biocatalitic properties of DNA; the structure of proteins; and other very recent problems in molecular biology. The text also provides a short course of modern topology intended for the broad audience of biologists and physicists. The authors are renowned specialists in their fields and some of the new results presented here are documented for the first time in monographic form.

Integration of Principles of Systems Biology and Radiation Biology: Toward Development of in silico Models to Optimize IUdR-Mediated Radiosensitization of DNA Mismatch Repair Deficient (Damage Tolerant) Human Cancers

International Nuclear Information System (INIS)

Kinsella, Timothy J.; Gurkan-Cavusoglu, Evren; Du, Weinan; Loparo, Kenneth A.

2011-01-01

Over the last 7 years, we have focused our experimental and computational research efforts on improving our understanding of the biochemical, molecular, and cellular processing of iododeoxyuridine (IUdR) and ionizing radiation (IR) induced DNA base damage by DNA mismatch repair (MMR). These coordinated research efforts, sponsored by the National Cancer Institute Integrative Cancer Biology Program (ICBP), brought together system scientists with expertise in engineering, mathematics, and complex systems theory and translational cancer researchers with expertise in radiation biology. Our overall goal was to begin to develop computational models of IUdR- and/or IR-induced base damage processing by MMR that may provide new clinical strategies to optimize IUdR-mediated radiosensitization in MMR deficient (MMR − ) “damage tolerant” human cancers. Using multiple scales of experimental testing, ranging from purified protein systems to in vitro (cellular) and to in vivo (human tumor xenografts in athymic mice) models, we have begun to integrate and interpolate these experimental data with hybrid stochastic biochemical models of MMR damage processing and probabilistic cell cycle regulation models through a systems biology approach. In this article, we highlight the results and current status of our integration of radiation biology approaches and computational modeling to enhance IUdR-mediated radiosensitization in MMR − damage tolerant cancers.

Fossil avian eggshell preserves ancient DNA

DEFF Research Database (Denmark)

Oskam, Charlotte L; Haile, James Seymour; McLay, Emma

2010-01-01

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful...... isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids......, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has...

DNA-Based Applications in Nanobiotechnology

Directory of Open Access Journals (Sweden)

Khalid M. Abu-Salah

2010-01-01

Full Text Available Biological molecules such as deoxyribonucleic acid (DNA have shown great potential in fabrication and construction of nanostructures and devices. The very properties that make DNA so effective as genetic material also make it a very suitable molecule for programmed self-assembly. The use of DNA to assemble metals or semiconducting particles has been extended to construct metallic nanowires and functionalized nanotubes. This paper highlights some important aspects of conjugating the unique physical properties of dots or wires with the remarkable recognition capabilities of DNA which could lead to miniaturizing biological electronics and optical devices, including biosensors and probes. Attempts to use DNA-based nanocarriers for gene delivery are discussed. In addition, the ecological advantages and risks of nanotechnology including DNA-based nanobiotechnology are evaluated.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

Science.gov (United States)

Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

2015-09-01

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Biological significance of the focus on DNA damage checkpoint factors remained after irradiation of ionizing radiation

International Nuclear Information System (INIS)

Yamauchi, Motohiro; Suzuki, Keiji

2005-01-01

This paper reviews recent reports on the focus formation and participation to checkpoint of (such phosphorylated (P-d) as below) ATM and H2AX, MDC1, 53BP1 and NBS1, and discusses their role in DNA damage checkpoint induction mainly around authors' studies. When the cell is irradiated by ionizing radiation, the subtype histone like H2AX is P-d and the formed focus', seen in the nucleus on immuno-fluorographic observation, represents the P-d H2AX at the damaged site of DNA. The role of P-d ATM (the product of causative gene of ataxia-telangiectasia mutation, a protein kinase) has been first shown by laser beam irradiation. Described are discussions on the roles and functions after irradiation in focus formation and DNA damage checkpoint of P-d H2AX (a specific histone product by the radiation like γ-ray as above), P-d ATM, MDC1 (a mediator of DNA damage check point protein 1), 53BP1, (a p53 binding protein) and NBS1 (the product of the causative gene of Nijmegen Breakage Syndrome). Authors have come to point out the remained focal size increase as implications of the efficient repair of damaged DNA, and the second cycled p53 accumulation, of tumor suppression. Thus evaluation of biological significance of these aspects, scarcely noted hitherto, is concluded important. (S.I.)

Quantitative estimation of the extent of alkylation of DNA following treatment of mammalian cells with non-radioactive alkylating agents

Energy Technology Data Exchange (ETDEWEB)

Snyder, R.D. (Univ. of Tennessee, Oak Ridge); Regan, J.D.

1981-01-01

Alkaline sucrose sedimentation has been used to quantitate phosphotriester formation following treatment of human cells with the monofunctional alkylating agents methyl and ethyl methanesulfonate. These persistent alkaline-labile lesions are not repaired during short-term culture conditions and thus serve as a useful and precise index of the total alkylation of the DNA.Estimates of alkylation by this procedure compare favorably with direct estimates by use of labeled alkylating agents.

A dynamic bead-based microarray for parallel DNA detection

International Nuclear Information System (INIS)

Sochol, R D; Lin, L; Casavant, B P; Dueck, M E; Lee, L P

2011-01-01

A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm 2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening

Principles of DNA architectonics: design of DNA-based nanoobjects

International Nuclear Information System (INIS)

Vinogradova, O A; Pyshnyi, D V

2012-01-01

The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

International Nuclear Information System (INIS)

Cai Xuwei; Fu Xiaolong; Yang Jian; Song Houyan

2005-01-01

Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- X TM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-X TM genome, which was transformed into E. coli to get recombinant Adeno-X TM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 10 11 TCID 50 /ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-X TM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

From biologically-inspired physics to physics-inspired biology From biologically-inspired physics to physics-inspired biology

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Kornyshev, Alexei A.

2010-10-01

The conference 'From DNA-Inspired Physics to Physics-Inspired Biology' (1-5 June 2009, International Center for Theoretical Physics, Trieste, Italy) that myself and two former presidents of the American Biophysical Society—Wilma Olson (Rutgers University) and Adrian Parsegian (NIH), with the support of an ICTP team (Ralf Gebauer (Local Organizer) and Doreen Sauleek (Conference Secretary)), have organized was intended to establish stronger links between the biology and physics communities on the DNA front. The relationships between them were never easy. In 1997, Adrian published a paper in Physics Today ('Harness the Hubris') summarizing his thoughts about the main obstacles for a successful collaboration. The bottom line of that article was that physicists must seriously learn biology before exploring it and even having an interpreter, a friend or co-worker, who will be cooperating with you and translating the problems of biology into a physical language, may not be enough. He started his story with a joke about a physicist asking a biologist: 'I want to study the brain. Tell me something about it!' Biologist: 'First, the brain consists of two parts, and..' Physicist: 'Stop. You have told me too much.' Adrian listed a few direct avenues where physicists' contributions may be particularly welcome. This gentle and elegantly written paper caused, however, a stormy reaction from Bob Austin (Princeton), published together with Adrian's notes, accusing Adrian of forbidding physicists to attack big questions in biology straightaway. Twelve years have passed and many new developments have taken place in the biologist-physicist interaction. This was something I addressed in my opening conference speech, with my position lying somewhere inbetween Parsegian's and Austin's, which is briefly outlined here. I will first recall certain precepts or 'dogmas' that fly in the air like Valkyries, poisoning those relationships. Since the early seventies when I was a first year Ph

uv photobiology: DNA damage and repair

International Nuclear Information System (INIS)

Sutherland, B.M.

1978-01-01

The following topics are discussed: targets that determine the fate of the cell when uv light interacts with a cell; comparison of action spectrum for a given biological effect with the absorption spectrum of different biological macromolecules; biological effects of damage to DNA; measurement of mutations; chemical damage to DNA; photoreactivation; role of pyrimidine dimers in induction of skin cancer by uv

DNA damage in plant herbarium tissue.

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Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

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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.

Quantitative biological measurement in Transmission Electron Tomography

International Nuclear Information System (INIS)

Mantell, Judith M; Verkade, Paul; Arkill, Kenton P

2012-01-01

It has been known for some time that biological sections shrink in the transmission electron microscope from exposure to the electron beam. This phenomenon is especially important in Electron Tomography (ET). The effect on shrinkage of parameters such as embedding medium or sample type is less well understood. In addition anisotropic area shrinkage has largely been ignored. The intention of this study is to explore the shrinkage on a number of samples ranging in thickness from 200 nm to 500 nm. A protocol was developed to determine the shrinkage in area and thickness using the gold fiducials used in electron tomography. In brief: Using low dose philosophy on the section, a focus area was used prior to a separate virgin study area for a series of known exposures on a tilted sample. The shrinkage was determined by measurements on the gold beads from both sides of the section as determined by a confirmatory tomogram. It was found that the shrinkage in area (approximately to 90-95% of the original) and the thickness (approximately 65% of the original at most) agreed with pervious authors, but that a lmost all the shrinkage was in the first minute and that although the direction of the in-plane shrinkage (in x and y) was sometimes uneven the end result was consistent. It was observed, in general, that thinner samples showed more percentage shrinkage than thicker ones. In conclusion, if direct quantitative measurements are required then the protocol described should be used for all areas studied.

Quantitative biological measurement in Transmission Electron Tomography

Science.gov (United States)

Mantell, Judith M.; Verkade, Paul; Arkill, Kenton P.

2012-07-01

It has been known for some time that biological sections shrink in the transmission electron microscope from exposure to the electron beam. This phenomenon is especially important in Electron Tomography (ET). The effect on shrinkage of parameters such as embedding medium or sample type is less well understood. In addition anisotropic area shrinkage has largely been ignored. The intention of this study is to explore the shrinkage on a number of samples ranging in thickness from 200 nm to 500 nm. A protocol was developed to determine the shrinkage in area and thickness using the gold fiducials used in electron tomography. In brief: Using low dose philosophy on the section, a focus area was used prior to a separate virgin study area for a series of known exposures on a tilted sample. The shrinkage was determined by measurements on the gold beads from both sides of the section as determined by a confirmatory tomogram. It was found that the shrinkage in area (approximately to 90-95% of the original) and the thickness (approximately 65% of the original at most) agreed with pervious authors, but that a lmost all the shrinkage was in the first minute and that although the direction of the in-plane shrinkage (in x and y) was sometimes uneven the end result was consistent. It was observed, in general, that thinner samples showed more percentage shrinkage than thicker ones. In conclusion, if direct quantitative measurements are required then the protocol described should be used for all areas studied.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

Science.gov (United States)

Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

DNA Replication Profiling Using Deep Sequencing.

Science.gov (United States)

Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

The intrinsic role of nanoconfinement in chemical equilibrium: evidence from DNA hybridization.

Science.gov (United States)

Rubinovich, Leonid; Polak, Micha

2013-05-08

Recently we predicted that when a reaction involving a small number of molecules occurs in a nanometric-scale domain entirely segregated from the surrounding media, the nanoconfinement can shift the position of equilibrium toward products via reactant-product reduced mixing. In this Letter, we demonstrate how most-recently reported single-molecule fluorescence measurements of partial hybridization of ssDNA confined within nanofabricated chambers provide the first experimental confirmation of this entropic nanoconfinement effect. Thus, focusing separately on each occupancy-specific equilibrium constant, quantitatively reveals extra stabilization of the product upon decreasing the chamber occupancy or size. Namely, the DNA hybridization under nanoconfined conditions is significantly favored over the identical reaction occurring in bulk media with the same reactant concentrations. This effect, now directly verified for DNA, can be relevant to actual biological processes, as well as to diverse reactions occurring within molecular capsules, nanotubes, and other functional nanospaces.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

Science.gov (United States)

Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

2016-10-01

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Processing of UV-induced DNA damage in diverse biological systems

International Nuclear Information System (INIS)

Galloway, A.M.

1992-01-01

A novel protocol has been developed allowing direct evaluation and accurate quantitation of UV lesions contained with both genomic DNA and the small oligonucleotides excised by a living cell during nucleotide excision repair. Using this methodology, the repair capacity of normal and UV-sensitive cells of human, Chinese hamster, and Escherichia coli origin, has been assessed. Several conclusions have been reached: (1) severage of the interpyrimidine phosphodiester linkage of cyclobutane dimers appears to be an evolutionarily conserved phenomenon; (2) the kinetics of cyclobutane dimer repair differ markedly from both (6-4) photoproduct and TA* lesion removal; (3) the ability to excise cyclobutane dimers is independent of (6-4) photoproduct repair capacity, suggesting that the lesions are not repaired/recognized by identical mechanisms; (4) fibroblast strains representing the eight xeroderma pigmentosum complementation groups each show a unique proficiency/deficiency to repair the different photolesions under study, implicating that a defect in a different locus underlies each genetic form of this disease; (5) the repair deficiency in UV-sensitive strains of trichothiodystrophy appears to be completely unrelated to that of non-complementing XP-D cells. To allow direct assessment of an IDP-altered photoproduct, substrates have been constructed which contain, at a defined dithymidine site, no lesion, a conventional cyclobutane dimer, or a cyclobutane dimer modified by severage of the intradimer phosphodiester bond. Bacteriophage T4 UV endonuclease has no activity towards a modified lesion, questioning the interpretation of experiments which utilize the strand-incising activity of this enzyme to monitor repair. Furthermore, although this altered lesion acts as a block to E. coli DNA polymerase I, it allows SP6 RNA polymerase to bypass the otherwise RNA polymerase-blocking lesion

A Cytomorphic Chip for Quantitative Modeling of Fundamental Bio-Molecular Circuits.

Science.gov (United States)

2015-08-01

We describe a 0.35 μm BiCMOS silicon chip that quantitatively models fundamental molecular circuits via efficient log-domain cytomorphic transistor equivalents. These circuits include those for biochemical binding with automatic representation of non-modular and loading behavior, e.g., in cascade and fan-out topologies; for representing variable Hill-coefficient operation and cooperative binding; for representing inducer, transcription-factor, and DNA binding; for probabilistic gene transcription with analogic representations of log-linear and saturating operation; for gain, degradation, and dynamics of mRNA and protein variables in transcription and translation; and, for faithfully representing biological noise via tunable stochastic transistor circuits. The use of on-chip DACs and ADCs enables multiple chips to interact via incoming and outgoing molecular digital data packets and thus create scalable biochemical reaction networks. The use of off-chip digital processors and on-chip digital memory enables programmable connectivity and parameter storage. We show that published static and dynamic MATLAB models of synthetic biological circuits including repressilators, feed-forward loops, and feedback oscillators are in excellent quantitative agreement with those from transistor circuits on the chip. Computationally intensive stochastic Gillespie simulations of molecular production are also rapidly reproduced by the chip and can be reliably tuned over the range of signal-to-noise ratios observed in biological cells.

Conformation-dependent DNA attraction.

Science.gov (United States)

Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

2014-06-21

Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

Optimized assembly and covalent coupling of single-molecule DNA origami nanoarrays.

Science.gov (United States)

Gopinath, Ashwin; Rothemund, Paul W K

2014-12-23

Artificial DNA nanostructures, such as DNA origami, have great potential as templates for the bottom-up fabrication of both biological and nonbiological nanodevices at a resolution unachievable by conventional top-down approaches. However, because origami are synthesized in solution, origami-templated devices cannot easily be studied or integrated into larger on-chip architectures. Electrostatic self-assembly of origami onto lithographically defined binding sites on Si/SiO2 substrates has been achieved, but conditions for optimal assembly have not been characterized, and the method requires high Mg2+ concentrations at which most devices aggregate. We present a quantitative study of parameters affecting origami placement, reproducibly achieving single-origami binding at 94±4% of sites, with 90% of these origami having an orientation within ±10° of their target orientation. Further, we introduce two techniques for converting electrostatic DNA-surface bonds to covalent bonds, allowing origami arrays to be used under a wide variety of Mg2+-free solution conditions.

Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

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Eveson J Paige

2006-08-01

Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

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Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Application of Near-Infrared Spectroscopy to Quantitatively Determine Relative Content of Puccnia striiformis f. sp. tritici DNA in Wheat Leaves in Incubation Period

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Yaqiong Zhao

2017-01-01

Full Text Available Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst is a devastating wheat disease worldwide. Potential application of near-infrared spectroscopy (NIRS in detection of pathogen amounts in latently Pst-infected wheat leaves was investigated for disease prediction and control. A total of 300 near-infrared spectra were acquired from the Pst-infected leaf samples in an incubation period, and relative contents of Pst DNA in the samples were obtained using duplex TaqMan real-time PCR arrays. Determination models of the relative contents of Pst DNA in the samples were built using quantitative partial least squares (QPLS, support vector regression (SVR, and a method integrated with QPLS and SVR. The results showed that the kQPLS-SVR model built with a ratio of training set to testing set equal to 3 : 1 based on the original spectra, when the number of the randomly selected wavelength points was 700, the number of principal components was 8, and the number of the built QPLS models was 5, was the best. The results indicated that quantitative detection of Pst DNA in leaves in the incubation period could be implemented using NIRS. A novel method for determination of latent infection levels of Pst and early detection of stripe rust was provided.

DNA damage and repair in plants

International Nuclear Information System (INIS)

Britt, A.B.

1996-01-01

The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses

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Woźniakowski Grzegorz

2012-03-01

Full Text Available Abstract Background Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV is infectious for geese and ducks while Muscovy duck parvovirus (MDPV infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR and loop-mediated isothermal amplification (LAMP were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. Results In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship

Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

International Nuclear Information System (INIS)

Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

2011-01-01

Highlights: → Mitochondrial dysfunction is central to many diseases of oxidative stress. → 95% of the mitochondrial genome is duplicated in the nuclear genome. → Dilution of untreated genomic DNA leads to dilution bias. → 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 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.

Exploring Phytoplankton Population Investigation Growth to Enhance Quantitative Literacy

Science.gov (United States)

Baumgartner, Erin; Biga, Lindsay; Bledsoe, Karen; Dawson, James; Grammer, Julie; Howard, Ava; Snyder, Jeffrey

2015-01-01

Quantitative literacy is essential to biological literacy (and is one of the core concepts in "Vision and Change in Undergraduate Biology Education: A Call to Action"; AAAS 2009). Building quantitative literacy is a challenging endeavor for biology instructors. Integrating mathematical skills into biological investigations can help build…

The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

International Nuclear Information System (INIS)

Chen, Chung H.

2004-01-01

The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

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.

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

2014-12-02

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. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

Science.gov (United States)

Nakamura, Shigetaka; Hashimoto, Hirokazu; Kobayashi, Satoshi; Fujimoto, Kenzo

2017-10-18

DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultraviolet absorption detection of DNA in gels

International Nuclear Information System (INIS)

Mahon, A.R.

1998-01-01

A method and apparatus for the detection and quantification of large fragments of unlabelled deoxyribonucleic acid (DNA) in agarose gels is presented. The technique is based on ultra-violet (UV) absorption by nucleotides. A deuterium lamp was used to illuminate regions of an electrophoresis gel. As DNA bands passed through the illuminated region of the gel the amount of UV light transmitted was reduced due to DNA absorption. Two detection systems were investigated. In the first system, synthetic chemical vapour deposition (CVD) diamond strip detectors were used to locate regions of DNA in the gels by detecting the transmitted light. CVD diamond has a high indirect band gap of 5.45 eV and is therefore sensitive to UV photons of wavelengths < 224 nm. A number of CVD diamond samples were characterised to investigate their suitability as detectors for this application. The detectors' quantum efficiency, UV response and time response were measured. DNA bands containing as little as 20 ng were detected by the diamond. In a second system, a deuterium lamp was used to illuminate individual sample lanes of an electrophoresis gel via an array of optical fibres. During electrophoresis the regions of DNA were detected with illumination at 260 nm, using a UV-sensitive charge coupled device (CCD). As the absorption coefficient of a DNA sample is approximately proportional to its mass, the technique is inherently quantitative. This system had a detection limit of 0.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. Using this detection technique, the DNA sample remains in its native state. The removal of carcinogenic dyes from the detection procedure greatly reduces associated biological hazards. (author)

Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

Science.gov (United States)

Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

2013-03-07

Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

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.

Quantitative Method of Measuring Metastatic Activity

Science.gov (United States)

Morrison, Dennis R. (Inventor)

1999-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated uroldnase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

Highly functionalized piperidines: Free radical scavenging, anticancer activity, DNA interaction and correlation with biological activity

Directory of Open Access Journals (Sweden)

Suvankar Das

2018-01-01

Full Text Available Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH and the anion radical superoxide (·O2−. The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to ·O2− scavenger was piperidine 10. In general, U251, MCF7, NCI/ADR-RES, NCI-H460 and HT29 cells were least sensitive to the tested compounds and all compounds were considerably more toxic to the studied cancer cell lines than to the normal cell line HaCaT. The binding mode of the compounds and ctDNA was preferably via intercalation. In addition, these results were confirmed based on theoretical studies. Finally, a linear and exponential correlation between interaction constant (Kb and GI50 for several human cancer cell was observed.

The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

International Nuclear Information System (INIS)

Oeck, Sebastian; Malewicz, Nathalie M.; Hurst, Sebastian; Rudner, Justine; Jendrossek, Verena

2015-01-01

The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci. For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience. The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone. The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the

A comparative approach for the investigation of biological information processing: An examination of the structure and function of computer hard drives and DNA

Science.gov (United States)

2010-01-01

Background The robust storage, updating and utilization of information are necessary for the maintenance and perpetuation of dynamic systems. These systems can exist as constructs of metal-oxide semiconductors and silicon, as in a digital computer, or in the "wetware" of organic compounds, proteins and nucleic acids that make up biological organisms. We propose that there are essential functional properties of centralized information-processing systems; for digital computers these properties reside in the computer's hard drive, and for eukaryotic cells they are manifest in the DNA and associated structures. Methods Presented herein is a descriptive framework that compares DNA and its associated proteins and sub-nuclear structure with the structure and function of the computer hard drive. We identify four essential properties of information for a centralized storage and processing system: (1) orthogonal uniqueness, (2) low level formatting, (3) high level formatting and (4) translation of stored to usable form. The corresponding aspects of the DNA complex and a computer hard drive are categorized using this classification. This is intended to demonstrate a functional equivalence between the components of the two systems, and thus the systems themselves. Results Both the DNA complex and the computer hard drive contain components that fulfill the essential properties of a centralized information storage and processing system. The functional equivalence of these components provides insight into both the design process of engineered systems and the evolved solutions addressing similar system requirements. However, there are points where the comparison breaks down, particularly when there are externally imposed information-organizing structures on the computer hard drive. A specific example of this is the imposition of the File Allocation Table (FAT) during high level formatting of the computer hard drive and the subsequent loading of an operating system (OS). Biological

A comparative approach for the investigation of biological information processing: an examination of the structure and function of computer hard drives and DNA.

Science.gov (United States)

D'Onofrio, David J; An, Gary

2010-01-21

The robust storage, updating and utilization of information are necessary for the maintenance and perpetuation of dynamic systems. These systems can exist as constructs of metal-oxide semiconductors and silicon, as in a digital computer, or in the "wetware" of organic compounds, proteins and nucleic acids that make up biological organisms. We propose that there are essential functional properties of centralized information-processing systems; for digital computers these properties reside in the computer's hard drive, and for eukaryotic cells they are manifest in the DNA and associated structures. Presented herein is a descriptive framework that compares DNA and its associated proteins and sub-nuclear structure with the structure and function of the computer hard drive. We identify four essential properties of information for a centralized storage and processing system: (1) orthogonal uniqueness, (2) low level formatting, (3) high level formatting and (4) translation of stored to usable form. The corresponding aspects of the DNA complex and a computer hard drive are categorized using this classification. This is intended to demonstrate a functional equivalence between the components of the two systems, and thus the systems themselves. Both the DNA complex and the computer hard drive contain components that fulfill the essential properties of a centralized information storage and processing system. The functional equivalence of these components provides insight into both the design process of engineered systems and the evolved solutions addressing similar system requirements. However, there are points where the comparison breaks down, particularly when there are externally imposed information-organizing structures on the computer hard drive. A specific example of this is the imposition of the File Allocation Table (FAT) during high level formatting of the computer hard drive and the subsequent loading of an operating system (OS). Biological systems do not have an

A comparative approach for the investigation of biological information processing: An examination of the structure and function of computer hard drives and DNA

Directory of Open Access Journals (Sweden)

D'Onofrio David J

2010-01-01

Full Text Available Abstract Background The robust storage, updating and utilization of information are necessary for the maintenance and perpetuation of dynamic systems. These systems can exist as constructs of metal-oxide semiconductors and silicon, as in a digital computer, or in the "wetware" of organic compounds, proteins and nucleic acids that make up biological organisms. We propose that there are essential functional properties of centralized information-processing systems; for digital computers these properties reside in the computer's hard drive, and for eukaryotic cells they are manifest in the DNA and associated structures. Methods Presented herein is a descriptive framework that compares DNA and its associated proteins and sub-nuclear structure with the structure and function of the computer hard drive. We identify four essential properties of information for a centralized storage and processing system: (1 orthogonal uniqueness, (2 low level formatting, (3 high level formatting and (4 translation of stored to usable form. The corresponding aspects of the DNA complex and a computer hard drive are categorized using this classification. This is intended to demonstrate a functional equivalence between the components of the two systems, and thus the systems themselves. Results Both the DNA complex and the computer hard drive contain components that fulfill the essential properties of a centralized information storage and processing system. The functional equivalence of these components provides insight into both the design process of engineered systems and the evolved solutions addressing similar system requirements. However, there are points where the comparison breaks down, particularly when there are externally imposed information-organizing structures on the computer hard drive. A specific example of this is the imposition of the File Allocation Table (FAT during high level formatting of the computer hard drive and the subsequent loading of an operating

DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study

Energy Technology Data Exchange (ETDEWEB)

Geisel, Dominik; Zimmermann, Elke; Rief, Matthias; Greupner, Johannes; Hamm, Bernd [Charite Medical School, Department of Radiology, Berlin (Germany); Laule, Michael; Knebel, Fabian [Charite Medical School, Department of Cardiology, Berlin (Germany); Dewey, Marc [Charite Medical School, Department of Radiology, Berlin (Germany); Charite, Institut fuer Radiologie, Berlin (Germany)

2012-08-15

To prospectively compare induced DNA double-strand breaks by cardiac computed tomography (CT) and conventional coronary angiography (CCA). 56 patients with suspected coronary artery disease were randomised to undergo either CCA or cardiac CT. DNA double-strand breaks were assessed in fluorescence microscopy of blood lymphocytes as indicators of the biological effects of radiation exposure. Radiation doses were estimated using dose-length product (DLP) and dose-area product (DAP) with conversion factors for CT and CCA, respectively. On average there were 0.12 {+-} 0.06 induced double-strand breaks per lymphocyte for CT and 0.29 {+-} 0.18 for diagnostic CCA (P < 0.001). This relative biological effect of ionising radiation from CCA was 1.9 times higher (P < 0.001) than the effective dose estimated by conversion factors would have suggested. The correlation between the biological effects and the estimated radiation doses was excellent for CT (r = 0.951, P < 0.001) and moderate to good for CCA (r = 0.862, P < 0.001). One day after radiation, a complete repair of double-strand breaks to background levels was found in both groups. Conversion factors may underestimate the relative biological effects of ionising radiation from CCA. DNA double-strand break assessment may provide a strategy for individualised assessments of radiation. (orig.)

Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids

International Nuclear Information System (INIS)

Cullum, M.E.; Zile, M.H.

1986-01-01

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C 18 reversed-phased analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that HPLC uv only conditions usually would not detect in the circulation and liver of rats under physiological conditions

Radiation physics, biophysics, and radiation biology. Progress report, December 1, 1992--November 30, 1993

Energy Technology Data Exchange (ETDEWEB)

Hall, E.J.; Zaider, M.

1993-05-01

Research at the Center for Radiological Research is a multidisciplenary blend of physics, chemistry and biology aimed at understanding the mechanisms involved in the health problems resulting from human exposure to ionizing radiations. The focus is increased on biochemistry and the application of the techniques of molecular biology to the problems of radiation biology. Research highlights of the program from the past year are described. A mathematical model describing the production of single-strand and double-strand breaks in DNA as a function radiation quality has been completed. For the first time Monte Carlo techniques have been used to obtain directly the spatial distribution of DNA moieties altered by radiation. This information was obtained by including the transport codes a realistic description of the electronic structure of DNA. We have investigated structure activity relationships for the potential oncogenicity of a new generation of bioreductive drugs that function as hypoxic cytotoxins. Experimental and theoretical investigation of the inverse dose rate effect, whereby medium LET radiations actually produce an c effect when the dose is protracted, is now at a point where the basic mechanisms are reasonably understood and the complex interplay between dose, dose rate and radiation quality which is necessary for the effect to be present can now be predicted at least in vitro. In terms of early radiobiological damage, a quantitative link has been established between basic energy deposition and locally multiply damaged sites, the radiochemical precursor of DNA double strand breaks; specifically, the spatial and energy deposition requirements necessary to form LMDs have been evaluated. For the first time, a mechanically understood ``biological fingerprint`` of high-LET radiation has been established. Specifically measurement of the ratio of inter-to intra-chromosomal aberrations produces a unique signature from alpha-particles or neutrons.

Biological Sexing of a 4000-Year-Old Egyptian Mummy Head to Assess the Potential of Nuclear DNA Recovery from the Most Damaged and Limited Forensic Specimens.

Science.gov (United States)

Loreille, Odile; Ratnayake, Shashikala; Bazinet, Adam L; Stockwell, Timothy B; Sommer, Daniel D; Rohland, Nadin; Mallick, Swapan; Johnson, Philip L F; Skoglund, Pontus; Onorato, Anthony J; Bergman, Nicholas H; Reich, David; Irwin, Jodi A

2018-03-01

High throughput sequencing (HTS) has been used for a number of years in the field of paleogenomics to facilitate the recovery of small DNA fragments from ancient specimens. Recently, these techniques have also been applied in forensics, where they have been used for the recovery of mitochondrial DNA sequences from samples where traditional PCR-based assays fail because of the very short length of endogenous DNA molecules. Here, we describe the biological sexing of a ~4000-year-old Egyptian mummy using shotgun sequencing and two established methods of biological sex determination (R X and R Y ), by way of mitochondrial genome analysis as a means of sequence data authentication. This particular case of historical interest increases the potential utility of HTS techniques for forensic purposes by demonstrating that data from the more discriminatory nuclear genome can be recovered from the most damaged specimens, even in cases where mitochondrial DNA cannot be recovered with current PCR-based forensic technologies. Although additional work remains to be done before nuclear DNA recovered via these methods can be used routinely in operational casework for individual identification purposes, these results indicate substantial promise for the retrieval of probative individually identifying DNA data from the most limited and degraded forensic specimens.

Biological Sexing of a 4000-Year-Old Egyptian Mummy Head to Assess the Potential of Nuclear DNA Recovery from the Most Damaged and Limited Forensic Specimens

Directory of Open Access Journals (Sweden)

Odile Loreille

2018-03-01

Full Text Available High throughput sequencing (HTS has been used for a number of years in the field of paleogenomics to facilitate the recovery of small DNA fragments from ancient specimens. Recently, these techniques have also been applied in forensics, where they have been used for the recovery of mitochondrial DNA sequences from samples where traditional PCR-based assays fail because of the very short length of endogenous DNA molecules. Here, we describe the biological sexing of a ~4000-year-old Egyptian mummy using shotgun sequencing and two established methods of biological sex determination (RX and RY, by way of mitochondrial genome analysis as a means of sequence data authentication. This particular case of historical interest increases the potential utility of HTS techniques for forensic purposes by demonstrating that data from the more discriminatory nuclear genome can be recovered from the most damaged specimens, even in cases where mitochondrial DNA cannot be recovered with current PCR-based forensic technologies. Although additional work remains to be done before nuclear DNA recovered via these methods can be used routinely in operational casework for individual identification purposes, these results indicate substantial promise for the retrieval of probative individually identifying DNA data from the most limited and degraded forensic specimens.

Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma.

Science.gov (United States)

Ogino, Shuji; Odze, Robert D; Kawasaki, Takako; Brahmandam, Mohan; Kirkner, Gregory J; Laird, Peter W; Loda, Massimo; Fuchs, Charles S

2006-09-01

Extensive gene promoter methylation in colorectal carcinoma has been termed the CpG island methylator phenotype (CIMP). Previous studies on CIMP used primarily methylation-specific polymerase chain reaction (PCR), which, unfortunately, may detect low levels of methylation that has little or no biological significance. Utilizing quantitative real-time PCR (MethyLight), we measured DNA methylation in a panel of 5 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 459 colorectal carcinomas obtained from 2 large prospective cohort studies. CIMP was defined as tumors that showed methylation in >or=4/5 promoters. CIMP was significantly associated with the presence of mucinous or signet ring cell morphology, marked Crohn's-like lymphoid reaction, tumor infiltrating lymphocytes, marked peritumoral lymphocytic reaction, tumor necrosis, tumor cell sheeting, and poor differentiation. All these features have previously been associated with microsatellite instability (MSI). Therefore, we divided the 459 colorectal carcinomas into 6 subtypes, namely, MSI-high (MSI-H)/CIMP, MSI-H/non-CIMP, MSI-low (MSI-L)/CIMP, MSI-L/non-CIMP, microsatellite stable/CIMP, and micro satellite sstable/non-CIMP. Compared with MSI-H/non-CIMP, MSI-H/CIMP was associated with marked tumor infiltrating lymphocytes, tumor necrosis, sheeting, and poor differentiation (all PCIMP, MSI-L/CIMP was associated with tumors that had CIMP. Both MSI and CIMP appear to play a role in the pathogenesis of specific morphologic patterns of colorectal carcinoma.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

Science.gov (United States)

Schmid, Volker J; Cremer, Marion; Cremer, Thomas

2017-07-01

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

An Introduction to DNA Fingerprinting.

Science.gov (United States)

Hepfer, Carol Ely; And Others

1993-01-01

Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

DNA adducts as molecular dosimeters

International Nuclear Information System (INIS)

Lucier, G.W.

1990-01-01

There is compelling evidence that DNA adducts play an important role in the actions of many pulmonary carcinogens. During the last ten years sensitive methods (antibodies and 32 P-postlabeling) have been developed that permit detection of DNA adducts in tissues of animals or humans exposed to low levels of some genotoxic carcinogens. This capability has led to approaches designed to more reliably estimate the shape of the dose-response curve in the low dose region for a few carcinogens. Moreover, dosimetry comparisions can, in some cases, be made between animals and humans which help in judging the adequacy of animal models for human risk assessments. There are several points that need to be considered in the evaluation of DNA adducts as a molecular dosimeter. For example, DNA adduct formation is only one of many events that are needed for tumor development and some potent carcinogens do not form DNA adducts; i.e., TCDD. Other issues that need to be considered are DNA adduct heterogeneity, DNA repair, relationship of DNA adducts to somatic mutation and cell specificity in DNA adduct formation and persistence. Molecular epidemiology studies often require quantitation of adducts in cells such as lymphocytes which may or may not be reliable surrogates for adduct concentrations in target issues. In summary, accurate quantitation of low levels of DNA adducts may provide data useful in species to species extrapolation of risk including the development of more meaningful human monitoring programs

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

Women are underrepresented in computational biology: An analysis of the scholarly literature in biology, computer science and computational biology.

Directory of Open Access Journals (Sweden)

Kevin S Bonham

2017-10-01

Full Text Available While women are generally underrepresented in STEM fields, there are noticeable differences between fields. For instance, the gender ratio in biology is more balanced than in computer science. We were interested in how this difference is reflected in the interdisciplinary field of computational/quantitative biology. To this end, we examined the proportion of female authors in publications from the PubMed and arXiv databases. There are fewer female authors on research papers in computational biology, as compared to biology in general. This is true across authorship position, year, and journal impact factor. A comparison with arXiv shows that quantitative biology papers have a higher ratio of female authors than computer science papers, placing computational biology in between its two parent fields in terms of gender representation. Both in biology and in computational biology, a female last author increases the probability of other authors on the paper being female, pointing to a potential role of female PIs in influencing the gender balance.

Women are underrepresented in computational biology: An analysis of the scholarly literature in biology, computer science and computational biology.

Science.gov (United States)

Bonham, Kevin S; Stefan, Melanie I

2017-10-01

While women are generally underrepresented in STEM fields, there are noticeable differences between fields. For instance, the gender ratio in biology is more balanced than in computer science. We were interested in how this difference is reflected in the interdisciplinary field of computational/quantitative biology. To this end, we examined the proportion of female authors in publications from the PubMed and arXiv databases. There are fewer female authors on research papers in computational biology, as compared to biology in general. This is true across authorship position, year, and journal impact factor. A comparison with arXiv shows that quantitative biology papers have a higher ratio of female authors than computer science papers, placing computational biology in between its two parent fields in terms of gender representation. Both in biology and in computational biology, a female last author increases the probability of other authors on the paper being female, pointing to a potential role of female PIs in influencing the gender balance.

Using electrophoretic mobility shift assays to measure equilibrium dissociation constants: GAL4-p53 binding DNA as a model system.

Science.gov (United States)

Heffler, Michael A; Walters, Ryan D; Kugel, Jennifer F

2012-01-01

An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K(D) ) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel; the DNA migrates more slowly when bound to a protein. To determine a K(D) the amount of unbound and protein-bound DNA in the gel is measured as the protein concentration increases. By performing this experiment, students will be introduced to making affinity measurements and gain experience in performing quantitative EMSAs. The experiment describes measuring the K(D) for the interaction between the chimeric protein GAL4-p53 and its DNA recognition site; however, the techniques are adaptable to other DNA binding proteins. In addition, the basic experiment described can be easily expanded to include additional inquiry-driven experimentation. © 2012 by The International Union of Biochemistry and Molecular Biology. Copyright © 2012 Wiley Periodicals, Inc.

Progress in quantitative X-ray microanalysis of frozen-hydrated bulk biological samples

International Nuclear Information System (INIS)

Marshall, A.T.

1988-01-01

The analysis of bulk frozen-hydrated biological samples has developed now to a level where practical application of the technique is possible. Provided the sample is carefully coated with a conductive metal, the development of a space charge capable of causing a significant distortion of the electron diffusion volume does not seem to occur, and analytical resolution can be conveniently held to approximately 2 micron (both depth and lateral resolution). Two valid quantitative methods are available, and two methods of determining dry weight fractions are also available. An area where further research could lead to improvement in analysis of frozen-hydrated bulk samples is in the investigation of fracturing methods. If fracture planes that were flat and reproducible could be easily obtained, some of the difficulties of analysing frozen-hydrated bulk samples would be considerably reduced

Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of pyrrolizidine alkaloid-induced tumorigenicity.

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Xia, Qingsu; Zhao, Yuewei; Von Tungeln, Linda S; Doerge, Daniel R; Lin, Ge; Cai, Lining; Fu, Peter P

2013-09-16

Pyrrolizidine alkaloid-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. The U.S. National Toxicology Program (NTP) classified riddelliine, a tumorigenic pyrrolizidine alkaloid, as "reasonably anticipated to be a human carcinogen" in the NTP 12th Report on Carcinogens in 2011. We previously determined that four DNA adducts were formed in rats dosed with riddelliine. The structures of the four DNA adducts were elucidated as (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine adducts (termed as DHP-dG-3 and DHP-dG-4) as the predominant adducts; and (ii) a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl)dehydrosupinidine adducts (termed as DHP-dA-3 and DHP-dA-4 adducts). In this study, we selected a nontumorigenic pyrrolizidine alkaloid, platyphylliine, a pyrrolizidine alkaloid N-oxide, riddelliine N-oxide, and nine tumorigenic pyrrolizidine alkaloids (riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, heliotrine, clivorine, and senkirkine) for study in animals. Seven of the nine tumorigenic pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, are liver carcinogens. At 8-10 weeks of age, female F344 rats were orally gavaged for 3 consecutive days with 4.5 and 24 μmol/kg body weight test article in 0.5 mL of 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed, livers were removed, and liver DNA was isolated for DNA adduct analysis. DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts were formed in the liver of rats treated with the individual seven hepatocarcinogenic pyrrolizidine alkaloids and riddelliine N-oxide. These DNA adducts were not formed in the liver of rats administered retronecine, the nontumorigenic pyrrolizidine alkaloid, platyphylliine, or vehicle control. These results indicate that this set of DNA adducts, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, is a common biological biomarker of

Building biological foundries for next-generation synthetic biology.

Science.gov (United States)

Chao, Ran; Yuan, YongBo; Zhao, HuiMin

2015-07-01

Synthetic biology is an interdisciplinary field that takes top-down approaches to understand and engineer biological systems through design-build-test cycles. A number of advances in this relatively young field have greatly accelerated such engineering cycles. Specifically, various innovative tools were developed for in silico biosystems design, DNA de novo synthesis and assembly, construct verification, as well as metabolite analysis, which have laid a solid foundation for building biological foundries for rapid prototyping of improved or novel biosystems. This review summarizes the state-of-the-art technologies for synthetic biology and discusses the challenges to establish such biological foundries.

Detection of base damage in DNA in human blood exposed to ionizing radiation at biologically relevant doses

International Nuclear Information System (INIS)

Loon, A.A.W.M. van; Lohman, P.H.M.; Groenendijk, R.H.; Schans, G.P. van der; Baan, R.A.

1991-01-01

The alkaline elution technique for the detection of DNA damage has been adapted to allow application on unlabelled blood cells. Both the induction and subsequent repair have been studied of two classes of DNA damage, viz. single-strand breaks and base damage recognized by the γ-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of base damage after in vitro exposure of full blood under aerobic conditions to biologically relevant doses of γ-rays (1.5-4.5 Gy). After a radiation dose of 3 Gy about 50% of the base damage was removed within 1.5 h of repair. Base damage could still be detected at 24h after exposure to 15 Gy. (author)

Quantitative assessment of the ion-beam irradiation induced direct damage of nucleic acid bases through FTIR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Huang, Qing, E-mail: huangq@ipp.ac.cn [Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences (China); University of Science and Technology of China, Hefei 230029, Anhui (China); Su, Xi; Yao, Guohua; Lu, Yilin; Ke, Zhigang; Liu, Jinghua; Wu, Yuejin; Yu, Zengliang [Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agriculture Engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences (China)

2014-07-01

Energetic particles exist ubiquitously in nature, and when they hit DNA molecules in organisms, they may induce critical biological effects such as mutation. It is however still a challenge to measure directly and quantitatively the damage imposed by the energetic ions on target DNA molecules. In this work we attempted to employ Fourier transformation infrared (FTIR) spectroscopy to assess the ion-induced direct damage of four nucleic acid bases, namely, thymine (T), cytosine (C), guanine (G), and adenine (A), which are the building blocks of DNA molecules. The samples were prepared as thin films, irradiated by argon ion-beams at raised ion fluences, and in the meantime measured by FTIR spectroscopy for the damage in a quasi-in-situ manner. It was found that the low-energy ion-beam induced radiosensitivity of the four bases shows the sequence G > T > C > A, wherein the possible mechanism was also discussed.

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

Science.gov (United States)

Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

2018-05-08

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mathematics of quantitative kinetic PCR and the application of standard curves.

Science.gov (United States)

Rutledge, R G; Côté, C

2003-08-15

Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

Science.gov (United States)

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Hands on Group Work Paper Model for Teaching DNA Structure, Central Dogma and Recombinant DNA

Science.gov (United States)

Altiparmak, Melek; Nakiboglu Tezer, Mahmure

2009-01-01

Understanding life on a molecular level is greatly enhanced when students are given the opportunity to visualize the molecules. Especially understanding DNA structure and function is essential for understanding key concepts of molecular biology such as DNA, central dogma and the manipulation of DNA. Researches have shown that undergraduate…

DNA Chemical discontinuities and their biological consequences

International Nuclear Information System (INIS)

Meneghini, R.

1978-01-01

The stability of genetic material is a relative concept since under several conditions there are structural changes in cellular DNA which unchain enzimatic processes leading to their own repair. Under certain circunstances the replication mechanism may arrive to a lesion before it be eliminated. It is known that most cells can replicate the injured DNA, but it is not known how this occurs. This mechanism is of great importance because there is strong evidence that mutations can be introduced in this process. Data are reviewed and discussed relating to the present stage of knowledge of this mechanism in bacteria and in mammal cells kept in culture. (M.A.) [pt

Bragg Curve, Biological Bragg Curve and Biological Issues in Space Radiation Protection with Shielding

Science.gov (United States)

Honglu, Wu; Cucinotta, F.A.; Durante, M.; Lin, Z.; Rusek, A.

2006-01-01

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LET gamma or X-rays, the presence of shielding does not always reduce the radiation risks for energetic charged particle exposure. Since the dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak, the Bragg curve does not necessarily represent the biological damage along the particle traversal since biological effects are influenced by the track structure of both primary and secondary particles. Therefore, the biological Bragg curve is dependent on the energy and the type of the primary particle, and may vary for different biological endpoints. To achieve a Bragg curve distribution, we exposed cells to energetic heavy ions with the beam geometry parallel to a monolayer of fibroblasts. Qualitative analyses of gamma-H2AX fluorescence, a known marker of DSBs, indicated increased clustering of DNA damage before the Bragg peak, enhanced homogenous distribution at the peak, and provided visual evidence of high linear energy transfer (LET) particle traversal of cells beyond the Bragg peak. A quantitative biological response curve generated for micronuclei (MN) induction across the Bragg curve did not reveal an increased yield of MN at the location of the Bragg peak. However, the ratio of mono-to bi-nucleated cells, which indicates inhibition in cell progression, increased at the Bragg peak location. These results, along with other biological concerns, show that space radiation protection with shielding can be a complicated issue.

Using Variable Precision Rough Set for Selection and Classification of Biological Knowledge Integrated in DNA Gene Expression

Directory of Open Access Journals (Sweden)

Calvo-Dmgz D.

2012-12-01

Full Text Available DNA microarrays have contributed to the exponential growth of genomic and experimental data in the last decade. This large amount of gene expression data has been used by researchers seeking diagnosis of diseases like cancer using machine learning methods. In turn, explicit biological knowledge about gene functions has also grown tremendously over the last decade. This work integrates explicit biological knowledge, provided as gene sets, into the classication process by means of Variable Precision Rough Set Theory (VPRS. The proposed model is able to highlight which part of the provided biological knowledge has been important for classification. This paper presents a novel model for microarray data classification which is able to incorporate prior biological knowledge in the form of gene sets. Based on this knowledge, we transform the input microarray data into supergenes, and then we apply rough set theory to select the most promising supergenes and to derive a set of easy interpretable classification rules. The proposed model is evaluated over three breast cancer microarrays datasets obtaining successful results compared to classical classification techniques. The experimental results shows that there are not significat differences between our model and classical techniques but it is able to provide a biological-interpretable explanation of how it classifies new samples.

Mesoscopic biology

Indian Academy of Sciences (India)

In this paper we present a qualitative outlook of mesoscopic biology where the typical length scale is of the order of nanometers and the energy scales comparable to thermal energy. Novel biomolecular machines, governed by coded information at the level of DNA and proteins, operate at these length scales in biological ...

A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

International Nuclear Information System (INIS)

Mahon, A.R.; MacDonald, J.H.; Mainwood, A.; Ott, R.J.

1999-01-01

A method and apparatus for the detection and quantification of large fragments of unlabelled nucleic acids in agarose gels is presented. The technique is based on ultraviolet (UV) absorption by nucleotides. A deuterium source illuminates individual sample lanes of an electrophoresis gel via an array of optical fibres. As DNA bands pass through the illuminated region of the gel the amount of UV light transmitted is reduced because of absorption by the DNA. During electrophoresis the regions of DNA are detected on-line using a UV-sensitive charge coupled device (CCD). As the absorption coefficient is proportional to the mass of DNA the technique is inherently quantitative. The mass of DNA in a region of the gel is approximately proportional to the integrated signal in the corresponding section of the CCD image. This system currently has a detection limit of less than 1.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. In addition the DNA sample remains in its native state. The removal of the carcinogenic dye from the detection procedure greatly reduces associated biological hazards. (author)

The progress of molecular biology in radiation research

International Nuclear Information System (INIS)

Wei Kang

1989-01-01

The recent progress in application of molecular biology techniques in the study of radiation biology is reviewed. The three sections are as follows: (1) the study of DNA damage on molecular level, (2) the molecular mechanism of radiation cell genetics, including chromosome abberation and cell mutation, (3) the study on DNA repair gene with DNA mediated gene transfer techniques

Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.

Science.gov (United States)

Millon, Laurence; Larosa, Fabrice; Lepiller, Quentin; Legrand, Faezeh; Rocchi, Steffi; Daguindau, Etienne; Scherer, Emeline; Bellanger, Anne-Pauline; Leroy, Joel; Grenouillet, Frederic

2013-05-01

The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

International Nuclear Information System (INIS)

Pinto, A. Viviana; Deodato, Elder L.; Cardoso, Janine S.; Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K.; Leitao, Alvaro C.; Padula, Marcelo de

2010-01-01

Although titanium dioxide (TiO 2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO 2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO 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 2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO 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 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 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 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.

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

NARCIS (Netherlands)

Buma, A.G.J.; Van Hannen, E.J.; Veldhuis, M.; Gieskes, W.W.C.

1996-01-01

The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp.

NARCIS (Netherlands)

Buma, A.G.J.; van Hannen, E.J; Veldhuis, M.J W; Gieskes, W.W C

The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

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)

Programmable chemical controllers made from DNA

Science.gov (United States)

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

2013-10-01

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

Molecular DNA Analysis in Forensic Identification.

Science.gov (United States)

Dumache, Raluca; Ciocan, Veronica; Muresan, Camelia; Enache, Alexandra

2016-01-01

Serological and biochemical identification methods used in forensics have several major disadvantages, such as: long time in processing biological sample and lack of sensitivity and specificity. In the last 30 years, DNA molecular analysis has become an important tool in forensic investigations. DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples. Forensic genetics, can provide information on the events which occurred at the crime scene or to supplement other methods of forensic identification. Currently, the methods used in identification are based on polymerase chain reaction (PCR) analyses. This method analyses the autosomal STRs, the Y-chromosome, and the mitochondrial DNA. Correlation of biological samples present at the crime scene with identification, selection, and the probative value factor is therefore the first aspect to be taken into consideration in the forensic genetic analysis. In the last decade, because of the advances in the field of molecular biology, new biomarkers such as: microRNAs (miR), messenger RNA (mRNA), and DNA methylation have been studied and proposed to be used in the forensic identifications of body fluids.

[Diagnostic significance of serum free DNA human telomerase reverse transcriptase quantitative determination on spinal cord injury].

Science.gov (United States)

Yang, M K; Tang, J; Xiang, Z; Zhang, X; Wang, J; Li, Z; Li, Y; Sheng, W B

2018-02-06

Objective: To investigate the relationship between the content of human telomerase reverse transcriptase (hTERT) and its clinical features in serum free DNA in patients with different degree of spinal cord injury. Methods: From December 2013 to December 2016, inpatients of the Central Hospital of Bazhong, Sichuan Province were enrolledand divided into the experimental group, the disease control group and the negative control group. For the experimental group: 46 patients with spinal cord injury were graded according to the criteria of the American Association of Spinal Cord Injury (ASIA), including 12 cases of grade A, 10 cases of grade B, 10 cases of grade C, 7 cases of grade D and 7 cases of grade E; for the disease control group: 15 patients with spinal fractures (without spinal cord injury) at the same period were included; and for the negative control group: 20 healthy adult volunteers aged 18-50 years were selected.Real-time fluorescence quantitative PCR and immunoblotting were performed to detect the content of hTERT in serum free DNA both in patients and healthy controls and to compare the difference between them. The results of the somatosensory evoked potential (SEP) of all patients were compared and analyzed.The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of hTERT content in serum free DNA in patients with spinal cord injury. Results: Comparison of serum free DNA hTERT content: in the experimental group, the serum free DNA hTERT content of grade A, B, C, D, E was (99.63±8.23), (76.24±4.37), (46.07±5.43), (16.30±0.95) and (15.74±1.12)μg/L, respectively.While it was (15.01±1.39)μg/L in the disease control group and (14.54±1.03)μg/L in the negative control group. The total difference was statistically significant between patients of each group and the control group ( F =857.917, P spinal cord injury has a certain guiding significance for the diagnosis of spinal cord injury and the degree of injury.

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.

The fusion of biology, computer science, and engineering: towards efficient and successful synthetic biology.

Science.gov (United States)

Linshiz, Gregory; Goldberg, Alex; Konry, Tania; Hillson, Nathan J

2012-01-01

Synthetic biology is a nascent field that emerged in earnest only around the turn of the millennium. It aims to engineer new biological systems and impart new biological functionality, often through genetic modifications. The design and construction of new biological systems is a complex, multistep process, requiring multidisciplinary collaborative efforts from "fusion" scientists who have formal training in computer science or engineering, as well as hands-on biological expertise. The public has high expectations for synthetic biology and eagerly anticipates the development of solutions to the major challenges facing humanity. This article discusses laboratory practices and the conduct of research in synthetic biology. It argues that the fusion science approach, which integrates biology with computer science and engineering best practices, including standardization, process optimization, computer-aided design and laboratory automation, miniaturization, and systematic management, will increase the predictability and reproducibility of experiments and lead to breakthroughs in the construction of new biological systems. The article also discusses several successful fusion projects, including the development of software tools for DNA construction design automation, recursive DNA construction, and the development of integrated microfluidics systems.

Quantitative analysis of biological fluids by electron probe and X ray spectrometry

International Nuclear Information System (INIS)

Girod, Chantal

1986-01-01

In order to know the kidney normal operation and to have an insight on cellular transport mechanisms and hormonal regulations at the nephron level, a technique based on the use of an electron probe has been developed for the elemental analysis of micro-volumes of biological fluids. This academic document reports applications of this technique on animals on which such fluids have been sampled at different levels of the nephron. As these samples are available in too small volumes to be dosed by conventional methods, they have been quantitatively analysed by using an electronic probe based analyser in order to determine concentrations of all elements with an atomic number greater than that of carbon. After a presentation of the implemented method and hardware, the author thus describes how an analysis is performed, and reports and discusses an example (analysis conditions, data acquisition, data processing, minimum detectable concentration, reasons for measurement scattering)

[Real-time quantification to analyze historical Colombian samples detecting a short fragment of hypervariable region II of mitochondrial DNA].

Science.gov (United States)

Pérez, Luz Adriana; Rodríguez, Freddy; Langebaek, Carl Henrik; Groot, Helena

2016-09-01

Unlike other molecular biology studies, the analysis of ancient DNA (aDNA) requires special infrastructure and methodological conditions to guarantee the quality of the results. One of the main authenticity criteria is DNA quantification, where quantitative real-time PCR is often used given its sensitivity and specificity. Nevertheless, the implementation of these conditions and methodologies to fulfill authenticity criteria imply higher costs. Objective: To develop a simple and less costly method for mitochondrial DNA quantification suitable for highly degraded samples. Materials and methods: The proposed method is based on the use of mini-primers for the specific amplification of short fragments of mitochondrial DNA. The subsequent purification of these amplified fragments allows a standard curve to be constructed with concentrations in accordance to the state of degradation of the samples. Results: The proposed method successfully detected DNA from ancient samples including bone remains and mummified tissue. DNA inhibitory substances were also detected. Conclusion: The proposed method represents a simpler and cost-effective way to detect low amounts of aDNA, and a tool to differentiate DNA-free samples from samples with inhibitory substances.

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

Science.gov (United States)

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

2012-01-01

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

Isotopes in molecular biology

International Nuclear Information System (INIS)

Goldfarb, P.S.G.

1988-01-01

The use of radioisotopes in molecular biology, with particular reference to the structure and functions of DNA, RNA and the cellular synthesis of proteins, is discussed. The use of labelled DNA and RNA in diagnostic techniques is presented. (U.K.)

Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

Science.gov (United States)

Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2012-01-01

N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

Science.gov (United States)

Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

2013-12-01

Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA-Controlled Assembly of Soft Nanoparticles

DEFF Research Database (Denmark)

Vogel, Stefan

2015-01-01

This book covers the emerging topic of DNA nanotechnology and DNA supramolecular chemistry in its broader sense. By taking DNA out of its biological role, this biomolecule has become a very versatile building block in materials chemistry, supramolecular chemistry and bio-nanotechnology. Many nove......-covalent systems, DNA origami, DNA based switches, DNA machines, and alternative structures and templates. This broad coverage is very appealing since it combines both the synthesis of modified DNA as well as designer concepts to successfully plan and make DNA nanostructures....

Improvement of Synthetic Biology Tools for DNA Editing

DEFF Research Database (Denmark)

Cavaleiro, Mafalda

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...... of gene circuits, synthesis of whole genomes and natural product discovery. In line with this, it is also described in this thesis how discovery of new cytochromes P450 (CYPs) from marine bacteria could benefit industrial processes....

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.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

Science.gov (United States)

Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.