Sample records for dna extraction step

  1. Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes.

    PubMed

    Ball, Shelley L; Armstrong, Karen F

    2008-04-01

    Early detection of economically important insects is critical to preventing their establishment as serious pests. To accomplish this, tools for rapid and accurate species identification are needed. DNA barcoding, using short DNA sequences as species "genetic identification tags," has already shown large potential as a tool for rapid and accurate detection of economically important insects. DNA extraction is the critical first step in generating DNA barcodes and can be a rate-limiting step in very large barcoding studies. Consequently, a DNA extraction method that is rapid, easy to use, cost-effective, robust enough to cope with range of qualities and quantities of tissue, and can be adapted to robotic systems will provide the best method for high-throughput production of DNA barcodes. We tested the performance of a new commercial kit (prepGEM), which uses a novel, streamlined approach to DNA extraction, and we compared it with two other commercial kits (ChargeSwitch and Aquapure), which differ in their method of DNA extraction. We compared performance of these kits by measuring percentage of polymerase chain reaction (PCR) success and mean PCR product yield across a variety of arthropod taxa, whichincluded freshly collected, ethanol-preserved, and dried specimens of different ages. ChargeSwitch and prepGEM performed equally well, but they outperformed Aquapure. prepGEM was much faster, easier to use, and cheaper than ChargeSwitch, but ChargeSwitch performed slightly better for older (> 5-yr-old) dried insect specimens. Overall, prepGEM may provide a highly streamlined method of DNA extraction for fresh, ethanol-preserved, and young, dried specimens, especially when adapted for high-throughput, robotic systems. PMID:18459420

  2. DNA Extraction

    NSDL National Science Digital Library

    Janice Stephens

    2011-01-01

    In this activity related to plant biotechnology, learners extract DNA from fruit to investigate how it looks and feels. The procedure is similar to what scientists have to do before they can use information contained in this DNA. This lesson guide includes procedure and discussion questions to help learners reflect on the process and purpose of DNA extraction. Modifications for younger learners are included in a related PDF (see related resources).

  3. Onion DNA Extraction

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can easily be extracted from onion cells using simple materials. Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  4. Yeast DNA Extraction

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can easily be extracted from yeast using simple materials. Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  5. Wheat Germ DNA Extraction

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can easily be extracted from wheat germ using simple materials. Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  6. DNA Extraction Virtual Lab

    NSDL National Science Digital Library

    2006-01-01

    This virtual lab from the Genetic Science Learning Center at the University of Utah provides a simple overview of DNA extraction, including what it's used for, illustrations, and an activity using cheek cells and laboratory equipment to isolate DNA. The lab is followed by a classroom activity that allows students and teachers to Extract DNA from Anything Living, using household items like spinach but not little sister's big toe.

  7. Thymus DNA Extractions

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can be extracted from a chunk of thymus (sweetbread) or liver. This experiment requires the use of a centrifuge (not included in cost of materials). Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  8. Exploring DNA Extraction Erica Butts

    E-print Network

    Perkins, Richard A.

    · Define DNA extraction · Downstream concerns for DNA extraction · Methods used to evaluate extraction into the extraction process and determine the amount recovered DNA Sources: #12;Applied Genetics Extraction MethodsApplied Genetics Exploring DNA Extraction Efficiency Erica Butts Research Biologist, Applied

  9. Fruitful DNA Extraction

    NSDL National Science Digital Library

    Karen Kalamuck

    2000-01-01

    In this lab activity, learners get to see and touch the genetic material they extract from the cells of a kiwi fruit - no high tech equipment required! After extraction and precipitation, learners will be able to collect the DNA with a wire hook. A facilitator's guide is included for helping educators run the activity, and background information is provided about what's going on, discussion questions, and ideas for inquiry. Biochemistry has never been so accessible - and fun!

  10. Lima Bean Bacteria DNA Extraction

    NSDL National Science Digital Library

    Lana Hays

    2009-01-01

    This laboratory exercise is designed to show learners how DNA can easily be extracted from lima bean bacteria. This experiment requires the use of a centrifuge (not included in cost of materials). Use this experiment to supplement any unit on genetics and to demonstrate how scientists study DNA. Adult supervision is recommended. This resource guide includes tips and suggestions for instructors as well as other DNA extraction experiments and a chart for learners to answer questions.

  11. DNA Extraction & Staging Laboratory (DESL)

    Cancer.gov

    As part of the Cancer Genomics Research Laboratory (CGR), the DNA Extraction and Staging Laboratory (DESL) located in Frederick, MD, is responsible for the preparation of samples for investigators at NCI's Division of Cancer Epidemiology and Genetics (DCEG).

  12. Extracting the Max From a DNA Extraction

    NSDL National Science Digital Library

    Edmund Marek

    2009-01-01

    Students of all ages get a thrill out of actually seeing clumps or strands of DNA. The Biotechnology/Bioinformatics Discovery! Project, a professional development workshop offered to science teachers, has always included a DNA-extraction activity. Over the course of four years, as the authors conducted these workshops for scores of teachers, they extended and refined the DNA-extraction activity to make it relevant to middle school students. Although the protocol for this exercise is on their project website along with teaching tips, they describe here the use of oral directions to give teachers many opportunities to interact with their students, and to assess how well students can follow directions and stay focused on the task.

  13. Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping,

    E-print Network

    California at Berkeley, University of

    for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from oneBack to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable

  14. MATERIALS AND METHODS 1) DNA extraction

    E-print Network

    Collins, Gary S.

    MATERIALS AND METHODS 1) DNA extraction · DNA was extracted from the ileo-cecal nodes of 475 Holstein cows from two herds using the Qiagen DNA extraction kit (Valencia, CA). 2) Map detection · Map and Jerome, Idaho, respectively. DNA was extracted from ileo-cecal lymph nodes using the Qiagen (Valencia, CA

  15. A Simple Automated DNA Extraction Method for Dried Blood Specimens Collected on Filter Paper

    Microsoft Academic Search

    Zhili Lin; Joseph G. Suzow; Jamie M. Fontaine; Edwin W. Naylor

    2005-01-01

    We developed a simple, inexpensive, and automated procedure for the extraction of genomic DNA from dried blood specimens (DBS) collected on filter paper. This DNA extraction method involves two simple steps. First, the DBS is treated with methanol. Genomic DNA is then extracted with Tris buffer in a heat incubation step. The use of common inexpensive chemicals such as methanol

  16. How to Extract DNA From Anything Living

    NSDL National Science Digital Library

    University of Utah

    2008-01-01

    In this genetics activity, learners discover how to extract DNA from green split peas. This resource guide includes a brief explanation of DNA and provides suggestions for ways to experiment with DNA extraction further.

  17. DNA BARCODING Tissue-direct PCR, a rapid and extraction-free method

    E-print Network

    DNA BARCODING Tissue-direct PCR, a rapid and extraction-free method for barcoding of ferns F-W. LI gametophytes and young sporophytes often provide too little material for DNA extraction and are particularly. in press). DNA extraction has always been the first step in the DNA barcoding process. However, in practice

  18. Automated DNA extraction from pollen in honey.

    PubMed

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. PMID:24295710

  19. DNA Extraction Techniques for Use in Education

    ERIC Educational Resources Information Center

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  20. Extracting DNA from a Banana

    NSDL National Science Digital Library

    Mark Gallo, Ph.D.

    2012-01-01

    Learners extract DNA from a banana. The procedure requires only basic lab equipment (i.e. beaker, test tube) and chemicals (i.e. liquid soap, meat tenderizer, ethanol). This activity is most appropriate for learners in grades 5-8. With slight modifications, this activity is appropriate for younger learners as well.

  1. A rapid and simple method for extracting yeast mitochondrial DNA

    Microsoft Academic Search

    Ali Gargouri; M. Curie

    1989-01-01

    A rapid method for the extraction of yeast mitochondrial DNA (mtDNA) is described. In comparison with previous methods, it simplifies several steps, does not require either the isolation of mitochondria or phenol treatment and is less time consuming. This protocol gives a high yield of pure mtDNA (50–120 µg from a 100-ml culture), which can be directly used in various

  2. Simplified buccal DNA extraction with FTA Elute Cards.

    PubMed

    Wolfgramm, Eldamária de Vargas; de Carvalho, Fernanda Magri; Aguiar, Vitor Rezende da Costa; Sartori, Mariana Penha De Nadai; Hirschfeld-Campolongo, Gabriela C R; Tsutsumida, Weslley M; Louro, Iúri Drumond

    2009-03-01

    DNA isolation is the initial step of most genetic studies, and ideally it should use a reliable and non-invasive method. Buccal samples are adequate for such purposes, being painless, easy to collect and a very reliable DNA source. FTA Elute Cards are relatively new on the market and are designed for rapid blood DNA extraction, in which DNA is solubilized in water, instead of attached to the paper matrix. In this study, we sought to test if FTA Elute Cards are suitable for buccal DNA extraction. Furthermore, several time and temperature conditions were analyzed in order to determine the best concentration/time ratio. Twenty-five different conditions were tested in quadruplicate and extracted DNA was quantified by real-time PCR. PMID:19215882

  3. DNA Extraction and Quantitation for Forensic Analysts

    NSDL National Science Digital Library

    This web site is part of the President's DNA Initiative and is devoted to the methodology for the extraction and quantification of DNA obtained from crime scene evidence. The site is designed as an on-line short course. The site identifies potential obstacles in the collection, extraction, and amplification of DNA. Extraction methods covered are organic, Chelex, and other extraction procedures. The site reviews inhibitors of the polymerase chain reaction (PCR) process and suggests methods for separating these inhibitors from the sample DNA. The advantages and disadvantages of commonly used methods for DNA are reviewed. The user must register and secure a readily obtainable password prior to entering the site.

  4. Rapid extraction and preservation of genomic DNA from human samples

    PubMed Central

    Kalyanasundaram, D.; Kim, J.-H.; Yeo, W.-H.; Oh, K.; Lee, K.-H.; Kim, M.-H.; Ryew, S.-M.; Ahn, S.-G.; Gao, D.; Cangelosi, G. A.; Chung, J.-H.

    2013-01-01

    Simple and rapid extraction of human genomic DNA remains a bottle neck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab- and saliva samples. DNA is attracted on to a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle 4 microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for one month was demonstrated for captured DNA, facilitating straightforward collection, delivery and handling of genomic DNA in an environment-friendly protocol. PMID:23307121

  5. Evaluation of DNA and RNA Extraction Methods

    Microsoft Academic Search

    C S Edwin Shiaw; M S Shiran; Y K Cheah; G C Tan; A R Sabariah; M Path

    2010-01-01

    SUMMARY This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method

  6. Evaluating the efficiency of DNA extraction methods from different substrates

    Microsoft Academic Search

    Timothy J. Verdon; R. John Mitchell; Roland A. H. van Oorschot

    The efficiency of extracting DNA directly from substrates varies according to a range of factors including the type of substrate and the extraction technique. Two routinely used DNA extraction methodologies (automated DNA IQ and manual Chelex) were texts on their efficiency to extract DNA from a range of blood volumes (0.1–30?L) on plastic and cotton. The efficiency of extracting DNA

  7. DNA profiling by different extraction methods

    Microsoft Academic Search

    A. Barbaro; N. Staiti; P. Cormaci; L. Saravo

    2004-01-01

    The aim of this study is to compare the efficiency of different validated methods for DNA extraction using commercial kits. DNA extraction was performed on fresh liquid blood, old bloodstains, cigarettes butts, semen stains and hairs. Samples were quantified by the AluQuant Human DNA Quantitation System. Amplification was carried out by GeneAmp 2400 and 9700 Thermal Cyclers using the AmpFlSTR

  8. DNA Basepair Step Deformability Inferred from Molecular Dynamics Simulations

    Microsoft Academic Search

    Filip Lankaš; Ji?í Šponer; Jörg Langowski; Thomas E. Cheatham III

    2003-01-01

    The sequence-dependent DNA deformability at the basepair step level was investigated using large-scale atomic resolution molecular dynamics simulation of two 18-bp DNA oligomers: d(GCCTATAAACGCCTATAA) and d(CTAGGTGGATGACTCATT). From an analysis of the structural fluctuations, the harmonic potential energy functions for all 10 unique steps with respect to the six step parameters have been evaluated. In the case of roll, three distinct

  9. Microcoding: the second step in DNA barcoding

    Microsoft Academic Search

    R. C. Summerbell; C. A. Lévesque; K. A. Seifert; M. Bovers; J. W. Fell; M. R. Diaz; T. Boekhout; G. S. de Hoog; J. A. Stalpers; P. W. Crous

    2005-01-01

    After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base

  10. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

    PubMed

    Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

    2011-08-01

    Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA. PMID:21815819

  11. Overcoming DNA extraction problems from carnivorous plants

    Microsoft Academic Search

    Andreas Fleischmann; Günther Heubl

    2009-01-01

    Fleischmann, A. & Heubl, G. 2009. Overcoming DNA extraction problems from carnivorous plants. Anales Jard. Bot. Madrid 66(2): 209-215. We tested previously published protocols for DNA isolation from plants with high contents of polyphenols and polysaccharides for several taxa of carnivorous plants. However, we did not get satisfying results with fresh or silica dried leaf tissue obtained from field collected

  12. A simple method to extract DNA from hair shafts using enzymatic laundry powder.

    PubMed

    Guan, Zheng; Zhou, Yu; Liu, Jinchuan; Jiang, Xiaoling; Li, Sicong; Yang, Shuming; Chen, Ailiang

    2013-01-01

    A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale. PMID:23922747

  13. Fern spore extracts can damage DNA.

    PubMed

    Simán, S E; Povey, A C; Ward, T H; Margison, G P; Sheffield, E

    2000-07-01

    The carcinogenicity of the vegetative tissues of bracken fern (Pteridium) has long been established. More recently, the carcinogenic effects of the spores of bracken have also been recognized. Both vegetative tissues and spores of bracken can induce adducts in DNA in animal tissues, but the possible genotoxic or carcinogenic effects of spores from fern species other than bracken are unknown. The single-cell gel electrophoresis ('comet') assay was used to investigate whether fern spores can cause DNA damage in vitro. Extracts of spores from six fern species were administered to cultured human premyeloid leukaemia (K562) cells. Spore extracts of five fern species: Anemia phyllitidis, Dicksonia antarctica, Pteridium aquilinum, Pteris vittata and Sadleria pallida, induced significantly more DNA strand breaks than those in the control groups. Only in one species, Osmunda regalis, was the effect no different from that in the control groups. Using extracts from A. phyllitidis and P. vittata, the extent of DNA damage was increased by increasing the original dose 10 times, whereas an experiment in which exposure times were varied suggested that the highest levels of strand breaks appear after 2 h exposure. Simultaneous incubation with human S9 liver enzyme mix ablated the damaging effect of the extracts. Our data show that fern spore extracts can cause DNA damage in human cells in vitro. Considering the strong correlation between DNA damage and carcinogenic events, the observations made in this report may well have some implications for human health. PMID:10883670

  14. A comparison of DNA extraction methods for food analysis

    Microsoft Academic Search

    Angela Di Pinto; VitoTony Forte; Maria Corsignano Guastadisegni; Carmela Martino; Francesco Paolo Schena; Giuseppina Tantillo

    2007-01-01

    In this paper, two DNA extraction and purification procedures for food analysis, Wizard® Magnetic DNA Purification for Food (Promega) and DNeasy® Tissue Kit (QIAGEN), have been compared concerning extraction efficiency, DNA purity and DNA suitability for amplification. The comparison of two extraction methods, in this study, has highlighted the efficiency of the Wizard® Magnetic DNA Purification approach for vegetable matrices

  15. Microscope Titration and Extraction of DNA from Liver.

    ERIC Educational Resources Information Center

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  16. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

    Microsoft Academic Search

    Anne Salonen; Janne Nikkilä; Jonna Jalanka-Tuovinen; Outi Immonen; Mirjana Rajili?-Stojanovi?; Riina A. Kekkonen; Airi Palva; Willem M. de Vos

    2010-01-01

    Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts

  17. Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction

    PubMed Central

    2014-01-01

    Background There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). Findings In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I’Etoile, France) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA was very successful. Conclusion QIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®. PMID:24447346

  18. Force steps during viral DNA packaging ?

    E-print Network

    Prashant K. Purohit; Jane' Kondev; Rob Phillips

    2003-09-22

    Biophysicists and structural biologists increasingly acknowledge the role played by the mechanical properties of macromolecules as a critical element in many biological processes. This change has been brought about, in part, by the advent of single molecule biophysics techniques that have made it possible to exert piconewton forces on key macromolecules and observe their deformations at nanometer length scales, as well as to observe the mechanical action of macromolecules such as molecular motors. This has opened up immense possibilities for a new generation of mechanical investigations that will respond to such measurements in an attempt to develop a coherent theory for the mechanical behavior of macromolecules under conditions where thermal and chemical effects are on an equal footing with deterministic forces. This paper presents an application of the principles of mechanics to the problem of DNA packaging, one of the key events in the life cycle of bacterial viruses with special reference to the nature of the internal forces that are built up during the DNA packaging process.

  19. Step-wise supercritical extraction of carbonaceous residua

    DOEpatents

    Warzinski, Robert P. (Venetia, PA)

    1987-01-01

    A method of fractionating a mixture containing high boiling carbonaceous material and normally solid mineral matter includes processing with a plurality of different supercritical solvents. The mixture is treated with a first solvent of high critical temperature and solvent capacity to extract a large fraction as solute. The solute is released as liquid from solvent and successively treated with other supercritical solvents of different critical values to extract fractions of differing properties. Fractionation can be supplemented by solute reflux over a temperature gradient, pressure let down in steps and extractions at varying temperature and pressure values.

  20. EXTRACTION OF GENOMIC DNA FROM PLANT TISSUE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three general methods that are commonly used to extract genomic DNA from plant tissue were reviewed. The principles, critical factors, and optimization procedures were discussed to help researcher to choose and modify a method according to their research purpose. Emphasis is given to a simple 96-we...

  1. DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps

    E-print Network

    extraction methods were compared to determine their efficacy in isolating DNA. Success of each method) in the gall-inhabiting complex. Keywords: cox 1, DNA extraction, minute insects, mtDNA barcoding Received 15DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps GUDRUN DITTRICH

  2. A comparison of five methods for extracting DNA from paucicellular clinical samples.

    PubMed

    Cler, Leslie; Bu, Dawei; Lewis, Cheryl; Euhus, David

    2006-01-01

    Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint measures. The methods included solid-phase DNA adsorption (QIAamp), sequential protein and DNA precipitation (Puregene), magnetic bead adsorption (Dynabeads), phenol-chloroform extraction, and single-step proteinase K digestion. In general, the performance of the three commercial kits was superior to either phenol-chloroform extraction or single-step proteinase K digestion. However, QIAamp and Puregene produced amplifiable DNA more frequently than Dynabeads for starting cell numbers <50,000. GAPDH Ct values for QIAamp extractions showed the greatest dynamic range and the best linearity across the range of starting cell numbers, but QIAamp was not statistically significantly superior to Puregene. Of the three commercial kits, Puregene is the least expensive. QIAamp and Puregene DNA extraction methods are well-suited for the preparation of paucicellular clinical samples for PCR-based assays. PMID:16516438

  3. COMPARISON OF DNA EXTRACTION METHODS ON DAIRY CONSTRUCTED WETLAND WASTEWATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct DNA extraction from environmental samples is a useful and culture-independent method for the examination of microbial diversity. To date, there is little information on the effectiveness of commercial DNA extraction kits on wastewater. We compared two commercial DNA extraction kits for amount...

  4. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    PubMed

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries. PMID:21519906

  5. Optimized microbial DNA extraction from diarrheic stools

    PubMed Central

    2012-01-01

    Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4%) non-lyophilised (Ct value 28.78?±?9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04?±?5.5); bacterial 16S rRNA was detected in 33/41 (80.5%) non-lyophilised (Ct value 28.11?±?5.9) versus 40/41 (97.6%) lyophilised aliquots (Ct value 24.94?±?6.6); and S. enterica was detected in 6/6 (100%) non-lyophilized and lyophilized aliquots (Ct value 26.98?±?4.55 and 26.16?±?4.97, respectively). S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p?=?0.00043) and Bacteria (p?=?0.015). Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea. PMID:23273000

  6. Optimising Bacterial DNA Extraction from Faecal Samples: Comparison of Three Methods

    PubMed Central

    Smith, Birgitte; Li, Nan; Andersen, Anders Schou; Slotved, Hans Christian; Krogfelt, Karen Angeliki

    2011-01-01

    Culture independent methods are used widely in diagnostic laboratories for infectious disease Isolation of genomic DNA from clinical samples is the first and important step in the procedure. Several procedures for extracting DNA from faecal samples have been described, including different mechanical cell disruptors. To our knowledge, the use of TissueLyser as a mechanical disruptor on faecal samples before DNA extraction has not been previously described. The purpose of the study was to implement a method for preparing faecal samples for optimal DNA extraction. Thus, three different procedures for extracting DNA from human faeces were compared. This was done either by using the mechanical disrupter by Mini BeadBeater 8, or the TissueLyser both followed by DNA purification using QIAamp DNA stool MiniKit, in comparison with DNA extractions using QIAamp DNA stool MiniKit without any prior mechanical disruption, according to manufacturer’s instructions. The obtained DNA from the three procedures was analysed by DGGE, and the number of bands was compared between each procedure. There was no significant difference between the numbers of bacterial bands obtained from DGGE when using a TissueLyser or Mini BeadBeater 8, so the two different mechanical cell disruptors can be used comparably when isolating bacterial DNA from faecal samples. The QIAamp DNA stool MiniKit alone resulted in a reduced number of bands compared to the two mechanical disruption methods. PMID:21643498

  7. Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

    SciTech Connect

    Damian, Luminita, E-mail: luminitadamian@microcal.eu.com [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France) [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France); Universite de Toulouse, UPS, IPBS, F-31077 Toulouse (France); IUB, School of Engineering and Science, D-28727 Bremen (Germany); Marty-Detraves, Claire, E-mail: claire.detraves@free.fr [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France) [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France); Universite de Toulouse, UPS, IPBS, F-31077 Toulouse (France); Winterhalter, Mathias [IUB, School of Engineering and Science, D-28727 Bremen (Germany)] [IUB, School of Engineering and Science, D-28727 Bremen (Germany); Fournier, Didier, E-mail: Didier.Fournier@ipbs.fr [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France) [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France); Universite de Toulouse, UPS, IPBS, F-31077 Toulouse (France); Paquereau, Laurent, E-mail: Laurent.Paquereau@ipbs.fr [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France) [CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse (France); Universite de Toulouse, UPS, IPBS, F-31077 Toulouse (France)

    2009-07-31

    Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K{sub d} = 3.62 {+-} 2.1 x 10{sup -8} M) or the RNA corresponding sequence (K{sub d} = 2.7 {+-} 0.82 x 10{sup -8} M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.

  8. Experiments in DNA Extraction and PCR Amplification from Bighorn Sheep Feces: the Importance of DNA Extraction Method

    Microsoft Academic Search

    J. D. Wehausen; R. R. RAMEY II; C. W. EPPS

    2004-01-01

    Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal

  9. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    PubMed

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. PMID:21820955

  10. A rapid and efficient assay for extracting DNA from fungi

    USGS Publications Warehouse

    Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.

    2002-01-01

    Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

  11. An Improved Protocol for DNA Extraction from Alkaline Soil and Sediment Samples for Constructing Metagenomic Libraries

    Microsoft Academic Search

    Digvijay Verma; T. Satyanarayana

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils\\u000a and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone\\u000a followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification\\u000a of DNA while minimizing the loss of DNA with respect

  12. Comparison of DNA extraction methods for multiplex polymerase chain reaction

    Microsoft Academic Search

    Triin Viltrop; Kaarel Krjutškov; Priit Palta; Andres Metspalu

    2010-01-01

    We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A260\\/280), PCR inhibition ratio, and mitochondrial DNA\\/genomic DNA

  13. Evaluation of Methods for the Extraction of DNA from Drinking Water Distribution System Biofilms

    PubMed Central

    Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L.; LeChevallier, Mark W.; Liu, Wen-Tso

    2012-01-01

    While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories. PMID:22075624

  14. Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions

    Microsoft Academic Search

    Peter R. Wielinga; Lianne de Heer; Astrid de Groot; Raditijo A. Hamidjaja; Geert Bruggeman; Kieran Jordan; Bart J. van Rotterdam

    2011-01-01

    The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based

  15. A fully automatable enzymatic method for DNA extraction from plant tissues

    PubMed Central

    Manen, Jean-François; Sinitsyna, Olga; Aeschbach, Lorène; Markov, Alexander V; Sinitsyn, Arkady

    2005-01-01

    Background DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA. Results Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species. Conclusion In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations. PMID:16269076

  16. DNA

    NSDL National Science Digital Library

    Jennifer Doherty

    In this activity, students extract DNA from their cheek cells and relate the steps in the procedure to the characteristics of cells and biological molecules. Students learn key concepts about the function of DNA during the intervals required for the extraction procedure. A second optional section develops student understanding of the fundamentals of DNA structure, function and replication; this section includes hands-on modeling of DNA replication. This activity, together with our activity, "From Gene to Protein - Transcription and Translation", can be used to teach the basic concepts of molecular biology.

  17. Composite system mediates two-step DNA uptake into Helicobacter pylori.

    PubMed

    Stingl, Kerstin; Müller, Stephanie; Scheidgen-Kleyboldt, Gerda; Clausen, Martin; Maier, Berenike

    2010-01-19

    The Gram-negative gastric pathogen Helicobacter pylori depends on natural transformation for genomic plasticity, which leads to host adaptation and spread of resistances. Here, we show that H. pylori takes up covalently labeled fluorescent DNA preferentially at the cell poles and that uptake is dependent on the type IV secretion system ComB. By titration of external pH and detection of accessibility of the fluorophor by protons, we localized imported fluorescent DNA in the periplasm. Single molecule analysis revealed that outer membrane DNA transport occurred at a velocity of 1.3 kbp x s(-1) and that previously imported DNA was reversibly extracted from the bacterium at pulling forces exceeding 23 pN. Thus, transport velocities were 10-fold higher than in Bacillus subtilis, and stalling forces were substantially lower. dsDNA stained with the intercalator YOYO-1 was transiently detected in the periplasm in wild-type H. pylori but was periplasmatically trapped in a mutant lacking the B. subtilis membrane-channel homolog ComEC. We conclude that H. pylori uses a two-step DNA uptake mechanism in which ComB transports dsDNA across the outer membrane at low force and poor specificity for DNA structure. Subsequently, Hp-ComEC mediates transport into the cytoplasm, leading to the release of the noncovalently bound DNA dye. Our findings fill the gap to propose a model for composite DNA uptake machineries in competent bacteria, all comprising the conserved ComEC channel for cytoplasmic membrane transport in combination with various transporters for access of external DNA to the cytoplasmic membrane. PMID:20080542

  18. Genomic DNA extraction method from pearl millet (Pennisetum glaucum) leaves

    Microsoft Academic Search

    Sghaier ZIDANI; Ali FERCHICHI; Mohamed CHAIEB

    2005-01-01

    DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both

  19. A Simple Automated Instrument for DNA Extraction in Forensic Casework

    Microsoft Academic Search

    Shawn A. Montpetit; Ian T. Fitch; Patrick T. O'Donnell

    2005-01-01

    ABSTRACT: The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples,in as few as 20min using magnetic,bead technology. The San Diego Police Department,Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence,and reference samples,in forensic casework. The BioRobot EZ1 was evaluated for use on

  20. Extraction of DNA from plant and fungus tissues in situ

    PubMed Central

    2012-01-01

    Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf?=?16,000×g), two battery-operated microcentrifuges (max rcf?=?5,000 and 3,000 ×g), and one manually-operated microcentrifuge (max rcf?=?120×g). Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide) DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt this method for genomic, metagenomic, transcriptomic and metabolomic projects using samples collected in situ. PMID:22672795

  1. One-stop Genomic DNA Extraction by Salicylic Acid Coated Magnetic Nanoparticles

    PubMed Central

    Zhou, Zhongwu; Kadam, Ulhas; Irudayaraj, Joseph

    2014-01-01

    Salicylic acid coated magnetic nanoparticles were prepared via a modified, one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by non-specific binding of the particles, as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared to traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally-friendly. PMID:23911528

  2. Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2014-01-01

    Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

  3. An easy, rapid, and inexpensive DNA extraction method, “one-minute DNA extraction,” for PCR in potato

    Microsoft Academic Search

    Kazuyoshi Hosaka

    2004-01-01

    A simple DNA extraction method was developed by modification of the SDSpotassiumacetate method (Dellaporta et al. 1983). It\\u000a can be completed in one minute and produced sufficient DNA for 500 RAPD or other conventional PCR amplifications.

  4. A rapid DNA extraction method for sugarcane and its relatives

    Microsoft Academic Search

    Rhonda J. Honeycutt; Bruno W. S. Sobral; Paul Keim; James E. Irvine

    1992-01-01

    A simple DNA extraction method based on CTAB precipitation was used to obtain DNA from members of the genusSaccharum and related species. DNA yields and purities were similar for allSaccharum species sampled. The method described here resulted in high quality total DNA suitable for polymerase chain reaction (PCR)-based\\u000a techniques as well as restriction endonuclease digestion, Southern hybridization, and DNA cycle-sequencing.

  5. Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR

    Microsoft Academic Search

    David N. Fredricks; Caitlin Smith; Amalia Meier

    2005-01-01

    The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to

  6. Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis

    Microsoft Academic Search

    Ming-Qi Deng; Dean O. Cliver

    1999-01-01

    A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient,

  7. Comparison of four DNA extraction methods for forensic application

    Microsoft Academic Search

    V. Bogas; F. Balsa; M. Carvalho; M. J. Anjos; M. F. Pinheiro; F. Corte-Real

    Challenging biological samples found in crime scenes are often brought to our lab. Several factors, such as degradation and the presence of inhibitors, can difficult the analysis of these samples. Chelating resin, silica membranes, silica-coated magnetic beads and paramagnetic resin were DNA extraction techniques used in this study. Our aim was to find out the DNA extraction method more suitable

  8. Combined lipid\\/DNA extraction method for environmental samples

    Microsoft Academic Search

    S. R. Kehrmeyer; B. M. Applegate; H. C. Pinkart; D. B. Hedrick; D. C. White; G. S. Sayler

    1996-01-01

    Previously, separate methods have been developed for the extraction and purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from the same environmental sample. This combined method is based on the Bligh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase

  9. An optimized DNA extraction protocol for benthic Didymosphenia geminata.

    PubMed

    Uyua, Noelia Mariel; Manrique, Julieta Marina; Jones, Leandro Roberto

    2014-09-01

    Didymosphenia geminata mats display few cells in relation to extracellular material and contain polysaccharides and heavy metals that interfere with molecular studies. We describe an optimized DNA extraction protocol that help to overcome these difficulties. Our protocol outperformed five previously described DNA extraction techniques. PMID:24946185

  10. A SIMPLE VERSATILE HIGH THROUGHPUT DNA EXTRACTION METHOD SUITABLE FOR PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR has become one of the most popular techniques in functional genomics. Projects in both forward and reverse genetics routinely require PCR amplification of thousands of samples. Processing samples to extract DNA of sufficient purity for PCR is often a limiting step. We have developed a simple ...

  11. Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts

    Microsoft Academic Search

    Marta Tortajada; Pedro Vicente Martínez-Culebras; Verónica Navarro; Honorato Monzó; Daniel Ramón

    2009-01-01

    Direct and sensitive PCR detection of contaminant microflora in cocoa extracts is affected by the quality of the template\\u000a DNA. This study compares the efficacy of five different commercial DNA extraction methods, selective enrichment broths and\\u000a use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artificially inoculated cocoa\\u000a extract samples. PCR-based methods

  12. Forensic evaluation of the QIAshredder\\/QIAamp DNA extraction procedure

    Microsoft Academic Search

    V. Castella; N. Dimo-Simonin; C. Brandt-Casadevall; P. Mangin

    2006-01-01

    The potential to recover genetic profiles from evidence samples has substantially increased since robust and sensitive amplification kits are commercially available. Nevertheless, even the best amplification kits cannot succeed when the extracted DNA is of poor quality. In this study we compared the efficiency of silica (QIAamp DNA Mini Kit), Chelex and Phenol–Chloroform (PC) based protocols to recover DNA from

  13. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  14. DNA, RNA, and Protein Extraction: The Past and The Present

    PubMed Central

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination. PMID:20011662

  15. Evaluation of DNA extraction methods suitable for PCR-based detection and genotyping of Clostridium botulinum.

    PubMed

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina; Skiby, Jeffrey E; De Medici, Dario

    2013-09-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. Eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin-producing clostridia were used to compare 4 DNA extraction procedures: Chelex(®) 100 matrix, Phenol-Cloroform-Isoamyl alcohol, NucliSENS(®) magnetic extraction kit, and DNeasy(®) Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy(®) Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However, Chelex(®) 100 matrix seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to DNeasy(®) Blood & Tissue kit, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results. PMID:23971807

  16. Bacterial and Archaeal DNA Extracted from Inoculated Experiments: Implication for the Optimization of DNA Extraction from Deep-Sea Basalts

    Microsoft Academic Search

    Hongmei Wang; Katrina J. Edwards

    2009-01-01

    Difficulties in efficient DNA extraction from deep-sea volcanic basalt, due to high metal concentration, complex organic matter, or sometimes the low biomass, have hampered the understanding of the significant biosphere both at and below the sea floor. In order to optimize the DNA extraction from basaltic rocks, sterilized basalts with different particle sizes and chemically synthesized goethite were inoculated with

  17. DNA walks one step at a time in electrophoresis

    NASA Astrophysics Data System (ADS)

    Guan, Juan; Wang, Bo; Granick, Steve

    2011-03-01

    Testing the classical view that in DNA gel electrophoresis, long polymer chains navigate through their gel environment via reptation, we reach a different conclusion: this driven motion proceeds by stick-slip. Our single-molecule experiments visualize fluorescent-labeled lambda-DNA, whose intramolecular conformations are resolved with 30 ms resolution using home-written software. Combining hundreds to thousands of trajectories under amplitudes of electric field ranging from zero to large, we quantify the full statistical distribution of motion with unprecedented statistics. Pauses are seen between steps of driven motion, probably reflecting that the chain is trapped inside the gel matrix. The pausing time is exponentially distributed and decreases with increasing electric field strength, suggesting that the jerky behavior is an activated process, facilitated by electric field. We propose a stretch-assisted mechanism: that the energy barrier to move through the gel environment is first overcome by a leading segment, the ensuing intramolecular stress from stretching causing lagging segments to recoil and follow along.

  18. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform

    PubMed Central

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-01-01

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ~36% for Chinese hamster ovary cells) and bacterial cells (up to ~45% for Salmonella typhimurium) that is comparable to the widely-used chemical lysis. The DNA extraction efficiency has dependence on both electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedure. PMID:23061629

  19. Efficient DNA extraction from hair shafts

    Microsoft Academic Search

    M. Almeida; E. Betancor; R. Fregel; N. M. Suárez; J. Pestano

    Hairs are common biological samples in crime scene investigation. However, most of this evidence is comprised of hair fragments without the root. As the major part of DNA is located in the root, hair shafts are usually problematic samples in forensic analysis. For these reasons, hair DNA typing is directed at mitochondrial DNA (mtDNA), which is present in high copy

  20. Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. Leaves.

    PubMed

    Sá, Olga; Pereira, José Alberto; Baptista, Paula

    2011-01-01

    Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds-namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 ?g/?L and the purity, evaluated by the ratio A(260)/A(280), was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR. PMID:21747730

  1. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

    PubMed Central

    Sá, Olga; Pereira, José Alberto; Baptista, Paula

    2011-01-01

    Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 ?g/?L and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR. PMID:21747730

  2. Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

    PubMed Central

    Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

    2014-01-01

    A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ?1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

  3. Hot-alkaline DNA extraction method for deep-subseafloor archaeal communities.

    PubMed

    Morono, Yuki; Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

    2014-03-01

    A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ?1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

  4. DNA extraction and amplification from Giemsa-stained blood smears.

    PubMed

    Yokota, M; Tatsumi, N; Tsuda, I; Yano, I

    1995-01-01

    DNA extraction was attempted from Giemsa-stained blood smears on glass slides that had been stored for several years. High molecular weight DNA bands were clearly visible after electrophoresis when DNA was extracted from specimens stored up to 2 years. Specimens more than 4 years old demonstrated a smeared pattern, suggesting degeneration of the DNA, but they could be rescued by PCR amplification using primers of HLA-DQA1 genes. The recovery could be pursued even in 11-year-old specimens. PMID:8587007

  5. Comparison of DNA Extraction Methods for Microbial Community Profiling with an Application to Pediatric Bronchoalveolar Lavage Samples

    Microsoft Academic Search

    Dana Willner; Joshua Daly; David Whiley; Keith Grimwood; Claire E. Wainwright; Philip Hugenholtz

    2012-01-01

    Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine

  6. [DNA extraction from body fluids of cadavers].

    PubMed

    Josephi, E; Braito, U; Tutsch-Bauer, E

    1990-01-01

    DNA of 1 ml Ascites, bile, liquor, pleural-exsudate, pericardial-fluid, synovial-fluid, urine and vitreous humor of sixteen bodies of either sex was isolated. Applying dot-blot-hybridisation black spots were obtained by using total human DNA as DNA-probe (A), if the origin is human. Our results showed, that body-fluids of dead persons contain human DNA, mainly. After rehybridisation with a sex-specific DNA-probe (Y) (pJA 1143) a determination of sex can be performed. PMID:2241771

  7. Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures.

    PubMed

    Votintseva, Antonina A; Pankhurst, Louise J; Anson, Luke W; Morgan, Marcus R; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W

    2015-04-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ?0.2 ng/?l of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools. PMID:25631807

  8. DNA extraction method from bones using Maxwell® 16.

    PubMed

    Karija Vlahovi?, Monika; Kubat, Milovan

    2012-09-01

    This paper describes the automated purification of DNA extracted from human bones using Maxwell® 16 bench top instrument. Analysis of nuclear short tandem repeats (STR) is invaluable in identification of human remains exhumed from mass graves in Croatia. Up to today 4683 skeletal remains have been recovered and for 897 human remains identity has not been determined. DNA has been extracted from 70% of all unidentified samples. For more than 90% of the samples nuclear STR profiles have been obtained using either organic phenol/chloroform method or silica-column purification for the extraction of DNA from bones or teeth. In order to evaluate a Maxwell® 16 DNA extraction performance 40 bone samples with different stage of decomposition were analyzed. The efficacy of manual silica based extraction and an automated purification was compared. The DNA yield per gram of starting material, removal of inhibitors and the quality of resulting STR profiles of the Maxwell extracts from duplicate amplifications were evaluated. The results show that Maxwell 16 platform can be used instead of manual DNA extraction procedures. PMID:22626613

  9. DNA extraction columns contaminated with murine sequences.

    PubMed

    Erlwein, Otto; Robinson, Mark J; Dustan, Simon; Weber, Jonathan; Kaye, Steve; McClure, Myra O

    2011-01-01

    Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques. PMID:21876752

  10. Comparison of DNA Extraction Methods in Analysis of Salivary Bacterial Communities

    PubMed Central

    Girard, Myriam; François, Patrice; Schrenzel, Jacques

    2013-01-01

    Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1–3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used. PMID:23844068

  11. PCR-DGGE detection of the bacterial community structure in the Inner Mongolia steppe with two different DNA extraction methods

    Microsoft Academic Search

    Zhou Xiaoqi; Wang Yanfen; Cai Ying; Huang Xiangzhong; Hao Yanbin; Tian Jianqing; Chai Tuanyao

    2007-01-01

    Compared with conventional methods, molecular biological technique, such as PCR-DGGE (denaturing gradient gel electrophoresis), is informative in examining the structure of the soil bacterial community through the extraction of microbial DNA from soil and generation of bacterial community profiles by PCR amplification of 16S rRNA genes. Extraction efficiency of soil microbial DNA is the most important step in these methods.

  12. Evaluation of DNA extraction methods from complex phototrophic biofilms

    Microsoft Academic Search

    Isabel Ferrera; Ramon Massana; Vanessa Balagué; Carles Pedrós-Alió; Olga Sánchez; Jordi Mas

    2010-01-01

    Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA

  13. A high-throughput DNA extraction method for barley seed

    Microsoft Academic Search

    Rebecka von Post; Lars von Post; Christophe Dayteg; Marie Nilsson; Brian P. Forster; Stine Tuvesson

    2003-01-01

    A non-destructive, quick DNA extraction method for barley seed is described. The method is simple and consists of drilling\\u000a out a sample from the seed, adding sodium hydroxide, heating in a microwave oven and neutralizing with Tris-HCl. The seed\\u000a DNA extract can be used directly for PCR with extra cycles added to the PCR programme compared to PCR programmes used

  14. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    PubMed Central

    Abusleme, Loreto; Hong, Bo-Young; Dupuy, Amanda K.; Strausbaugh, Linda D.; Diaz, Patricia I.

    2014-01-01

    Background and objective The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies. PMID:24778776

  15. Dellaporta DNA Extraction Citation: Stephen L. Dellaporta,Jonathan Wood , James B. Hicks. A plant DNA

    E-print Network

    Wurtele, Eve Syrkin

    1 Dellaporta DNA Extraction Citation: Stephen L. Dellaporta,Jonathan Wood , James B. Hicks. A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1983, Volume 1, Issue 4, pp 19 supernatant and lightly dry DNA pellets by inverting the tubes on paper towels for 10 min. #12;4 12

  16. EXTRACTION AND PURIFICATION OF MICROBIAL DNA FROM SEDIMENTS

    EPA Science Inventory

    A new method for the isolation of intracellular and extracellular DNA from a range of sediment types has been developed. The method is based upon the direct lysis of cells in the sediment, extraction of released DNA from the sediments and its subsequent purification by CsC1-EtBr ...

  17. Ancient DNA: Extraction, Characterization, Molecular Cloning, and Enzymatic Amplification

    Microsoft Academic Search

    Svante Paabo

    1989-01-01

    Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest

  18. Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

    PubMed Central

    Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J.; Waghorn, Garry C.; Janssen, Peter H.

    2013-01-01

    Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided. PMID:24040342

  19. Comparison of DNA extraction methods for identification of human remains

    Microsoft Academic Search

    Felicity Pagan; Cindy Lim; Mojca Keglovic; Dennis McNevin

    2011-01-01

    After mass disasters, where the bodies of victims have been degraded, soft tissues are often completely lost, with the result that bones and teeth are all that remain for identification. The aim of this study was to investigate three DNA extraction methods that are currently used with degraded bones in forensic contexts: a silica-based extraction protocol used by the International

  20. Comparison of DNA extraction methods for identification of human remains

    Microsoft Academic Search

    Felicity Pagan; Cindy Lim; Mojca Keglovic; Dennis McNevin

    2012-01-01

    After mass disasters, where the bodies of victims have been degraded, soft tissues are often completely lost, with the result that bones and teeth are all that remain for identification. The aim of this study was to investigate three DNA extraction methods that are currently used with degraded bones in forensic contexts: a silica-based extraction protocol used by the International

  1. Stepped electrophoresis for movement and concentration of DNA

    DOEpatents

    Miles, Robin R.; Wang, Amy Wei-Yun; Mariella Jr., Raymond P.

    2005-03-15

    A fluidic channel patterned with a series of thin-film electrodes makes it possible to move and concentrate DNA in a fluid passing through the fluidic channel. The DNA has an inherent negative charge and by applying a voltage between adjacent electrodes the DNA is caused to move. By using a series of electrodes, when one electrode voltage or charge is made negative with respect to adjacent electrodes, the DNA is repelled away from this electrode and attached to a positive charged electrode of the series. By sequentially making the next electrode of the series negative, the DNA can be moved to and concentrated over the remaining positive electrodes.

  2. Purification of crime scene DNA extracts using centrifugal filter devices

    PubMed Central

    2013-01-01

    Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

  3. Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA

    E-print Network

    Ljubljana, University of

    . Differ- ent pDNA extraction methods have been described; among them is alkaline lysis, currently the most cost of production. We compared the con- centration of extracted pDNA when two methods for extracting pDNA lysis, suggesting the use of electro- poration as a potentially superior method for extracting pDNA from

  4. Extracting biological knowledge from DNA sequences

    SciTech Connect

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

    1996-12-31

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

  5. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    PubMed

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. PMID:23872000

  6. New DNA Extraction Methods for Casework Evidence

    Microsoft Academic Search

    Martha F. Burger; Todd W. Bille; James W. Schumm

    To ease the national backlog of crime scene evidence requiring DNA analysis, we have adopted methods that support high throughput processing of casework samples. In recent years, we have developed a new device for simplified reference sample collection, new protocols for a rapid pre-screen for Y chromosome DNA, and a microplate-compatible human-specific quantification method (Fox et al., 2003). Here we

  7. Field preservation and DNA extraction methods for intestinal microbial diversity analysis in earthworms.

    PubMed

    Thakuria, Dwipendra; Schmidt, Olaf; Liliensiek, Ann-Kathrin; Egan, Damian; Doohan, Fiona M

    2009-03-01

    We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P<0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P> or =0.05), the former method yielded >1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P<0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT- as compared to the SDS-based method (P<0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R=0.76, P<0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P<0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R=0.79 and 0.83 for bacteria and fungi, respectively; P<0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates. PMID:19038293

  8. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    PubMed

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. PMID:22310206

  9. Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

    Microsoft Academic Search

    Luminita Damian; Claire Marty-Detraves; Mathias Winterhalter; Didier Fournier; Laurent Paquereau

    2009-01-01

    Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants

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

    PubMed

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

    2011-03-01

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

  11. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  12. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis

    PubMed Central

    Kapustin, D. V.; Prostyakova, A. I.; Alexeev, Ya. I.; Varlamov, D. A.; Zubov, V. P.; Zavriev, S. K.

    2014-01-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  13. Evaluation of the ISO Standard 11063 DNA Extraction Procedure for Assessing Soil Microbial Abundance and Community Structure

    PubMed Central

    Griffiths, Robert; Dequiedt, Samuel; Lelievre, Mélanie; Regnier, Tiffanie; Nowak, Virginie; Bailey, Mark; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Mougel, Christophe; Ranjard, Lionel

    2012-01-01

    Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities. PMID:22984486

  14. Evaluation of the ISO standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure.

    PubMed

    Plassart, Pierre; Terrat, Sébastien; Thomson, Bruce; Griffiths, Robert; Dequiedt, Samuel; Lelievre, Mélanie; Regnier, Tiffanie; Nowak, Virginie; Bailey, Mark; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Mougel, Christophe; Ranjard, Lionel

    2012-01-01

    Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities. PMID:22984486

  15. DNA mismatch repair detected in human cell extracts.

    PubMed

    Glazer, P M; Sarkar, S N; Chisholm, G E; Summers, W C

    1987-01-01

    A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone. PMID:3031461

  16. Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

    Microsoft Academic Search

    F. M. Lakay; A. Botha; B. A. Prior

    2007-01-01

    Aim: To establish a rapid, improved soil environmental DNA extraction and purification protocol. Methods and Results: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of suc- cess. Bead beating extraction gave significantly higher DNA yields than micro- wave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing

  17. A more consistent method for extracting and amplifying DNA from bee wings

    E-print Network

    A more consistent method for extracting and amplifying DNA from bee wings Elaine M. GOULD, Michelle reaction (PCR) amplification. Here, we describe an improved method for extracting DNA from bee wings using providing only a small sample of tissue and DNA. One method that is commonly used to extract DNA from bee

  18. TECHNICAL ADVANCES A rapid column-based ancient DNA extraction method for

    E-print Network

    Reich, David

    TECHNICAL ADVANCES A rapid column-based ancient DNA extraction method for increased sample-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples a method that combines the high DNA yield of batch-based silica extraction with the time

  19. Strawberry DNA Extraction 2009 1 Minority Science Programs School of Biological Sciences University of California, Irvine

    E-print Network

    Rose, Michael R.

    Strawberry DNA Extraction 2009 1 Minority Science Programs ­ School regular household chemicals to extract DNA from strawberries and other fruits. Each chemical has a specific purpose in the process of extracting DNA. Scientists use a very similar process to isolate DNA

  20. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment.

    PubMed Central

    Moré, M I; Herrick, J B; Silva, M C; Ghiorse, W C; Madsen, E L

    1994-01-01

    This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8017936

  1. Real-time PCR evaluation of seven DNA extraction methods for the purpose of GMO analysis

    Microsoft Academic Search

    Mohamad Ghazali; Jalan Sultan

    Successful DNA amplification is vital for the detection of specific DNA targets in feeds, and this in return depends on the ability of DNA extraction methods to produce good quality DNA. In this study, seven methods were compared for DNA extraction from feeds using quantitative polymerase chain reaction (PCR) of single copy maize ( Zea mays ) endogenous hmg (high

  2. Extraction of DNA from dried blood in the diagnosis of congenital CMV infection.

    PubMed

    de Vries, Jutte J C; Barbi, Maria; Binda, Sandro; Claas, Eric C J

    2012-01-01

    Viral DNA detection in dried blood spotted on filter paper, dried blood spots (DBS), is valuable in the diagnosis of viral infections, with at the moment congenital cytomegalovirus (CMV) being the most common application. CMV detection in clinical samples taken within the first 2-3 weeks after birth differentiates congenital CMV infection from the in general harmless postnatal acquired cytomegalovirus infection. DBS render the possibility to diagnose congenital CMV infection retrospectively, e.g., when late-onset hearing loss, the most frequently encountered symptom of congenital CMV infection, becomes manifest. Additionally, CMV DNA detection in DBS can be of usage in recently advocated newborn screening on congenital CMV infection. The procedure of CMV DNA detection in DBS consists of two separate steps: (1) DNA extraction from the DBS, followed by (2) CMV DNA amplification. Here, we describe two efficient methods for the extraction of DNA from DBS. Sensitivity, specificity, and applicability of the methods for high-throughput usage are discussed. PMID:22782817

  3. Direct Extraction and Amplification of DNA from Soil.

    ERIC Educational Resources Information Center

    Trevors, Jack T.; Leung, K.

    1998-01-01

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

  4. A simple procedure for the extraction of trypanosome DNA and host protein from dried blood meal residues of haematophagous Diptera

    Microsoft Academic Search

    R. Boid; T. W. Jones; A. Munro

    1999-01-01

    A two step elution method is described for the extraction of host serum proteins and trypanosome DNA from a single dried insect gut smear preparation. The first low temperature elution yields material suitable for use in ELISA to determine the host species on which the fly last fed while the results of the second, high temperature, elution can be used

  5. Development of microfluidic modules for DNA purification via phenol extraction and analyte concentration using transverse electrokinetics

    NASA Astrophysics Data System (ADS)

    Morales, Mercedes C.

    In this work, microfluidic platforms have been designed and evaluated to demonstrate microscale DNA purification via organic (phenol) extraction as well as analyte trapping and concentration using a transverse electrokinetic force balance. First, in order to evaluate DNA purification via phenol extraction in a microdevice, an aqueous phase containing protein and DNA and an immiscible receiving organic phase were utilized to evaluate microfluidic DNA extraction under both stratified and droplet-based flow conditions using a serpentine microfluidic device. The droplet based flow resulted in a significant improvement of protein partitioning from the aqueous phase due to the flow recirculation inside each droplet improving material convective transport into the organic phase. The plasmid recovery from bacterial lysates using droplet-based flow was high (>92%) and comparable to the recovery achieved using commercial DNA purification kits and standard macroscale phenol extraction. Second, a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields. Negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main channel where transverse electric fields were applied. Experimental results demonstrated that charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility and high electrophoretic mobility condition (high ionic strength buffer) or migrated towards an equilibrium position within the channel when both electroosmotic mobility and electrophoretic mobility are high (low ionic strength buffer). The different behaviors are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. The miniaturization of DNA phenol extraction and the novel electrokinetic trapping techniques presented in this research are the initial steps towards an efficient DNA sample preparation chip which could be integrated with post-extraction DNA manipulations for genomic analysis modules such as capillary electrophoretic separations.

  6. DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR.

    PubMed

    Hawash, Yousry

    2014-06-01

    PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ? 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis. PMID:25031466

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

    PubMed

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

    2014-12-01

    To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method. PMID:25381609

  8. One-step synthesis of silver nanoparticles, nanorods, and nanowires on the surface of DNA network.

    PubMed

    Wei, Gang; Zhou, Hualan; Liu, Zhiguo; Song, Yonghai; Wang, Li; Sun, Lanlan; Li, Zhuang

    2005-05-12

    Here, we describe a one-step synthesis of silver nanoparticles, nanorods, and nanowires on DNA network surface in the absence of surfactant. Silver ions were first adsorbed onto the DNA network and then reduced in sodium borohydride solution. Silver nanoparticles, nanorods, and nanowires were formed by controlling the size of pores of the DNA network. The diameter of the silver nanoparticles and the aspect ratio of the silver nanorods and nanowires can be controlled by adjusting the DNA concentration and reduction time. PMID:16852035

  9. Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples.

    PubMed

    Greenspoon, Susan A; Ban, Jeffrey D; Sykes, Karen; Ballard, Elizabeth J; Edler, Shelley S; Baisden, Melissa; Covington, Brian L

    2004-01-01

    Robotic systems are commonly utilized for the extraction of database samples. However, the application of robotic extraction to forensic casework samples is a more daunting task. Such a system must be versatile enough to accommodate a wide range of samples that may contain greatly varying amounts of DNA, but it must also pose no more risk of contamination than the manual DNA extraction methods. This study demonstrates that the BioMek 2000 Laboratory Automation Workstation, used in combination with the DNA IQ System, is versatile enough to accommodate the wide range of samples typically encountered by a crime laboratory. The use of a silica coated paramagnetic resin, as with the DNA IQ System, facilitates the adaptation of an open well, hands off, robotic system to the extraction of casework samples since no filtration or centrifugation steps are needed. Moreover, the DNA remains tightly coupled to the silica coated paramagnetic resin for the entire process until the elution step. A short pre-extraction incubation step is necessary prior to loading samples onto the robot and it is at this step that most modifications are made to accommodate the different sample types and substrates commonly encountered with forensic evidentiary samples. Sexual assault (mixed stain) samples, cigarette butts, blood stains, buccal swabs, and various tissue samples were successfully extracted with the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System, with no evidence of contamination throughout the extensive validation studies reported here. PMID:14979341

  10. A simple method for DNA extraction from marine bacteria that produce extracellular materials

    Microsoft Academic Search

    Y. K Lee; H. W Kim; C. L Liu; H. K Lee

    2003-01-01

    We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified

  11. Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils

    Microsoft Academic Search

    G Lloyd-Jones; D. W. F Hunter

    2001-01-01

    The direct extraction of DNA from different New Zealand soils using three simple and rapid methods has revealed that poor recovery of DNA during extraction was influenced by extraction procedure and also edaphic factors. Clay mineralogy and purification strategies required to remove compounds, such as humic acids that are inhibitory to DNA analysis, can significantly reduce yields and are dependant

  12. Evaluation of DNA extraction methods from green and roasted coffee beans

    Microsoft Academic Search

    Stelios Spaniolas; Maroussa Tsachaki; Malcolm J. Bennett; Gregory A. Tucker

    2008-01-01

    This paper describes a generic approach to the evaluation of DNA extraction methodology using green and roasted coffee beans as a model commodity. The use of Design-Expert® software was used in the design of experimental work to compare and optimize yields using a variety of commercial DNA extraction kits. The quality of the extracted DNA in terms of PCR inhibitor

  13. Genomic DNA Extraction from Cells by Electroporation on an Integrated Microfluidic Platform

    E-print Network

    Lu, Chang

    -21 Chemical lysis methods are most commonly used for DNA extraction either on-chip22-27 or off-chip.28Genomic DNA Extraction from Cells by Electroporation on an Integrated Microfluidic Platform Tao. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from

  14. Field preservation and DNA extraction methods for intestinal microbial diversity analysis in earthworms

    Microsoft Academic Search

    Dwipendra Thakuria; Olaf Schmidt; Ann-Kathrin Liliensiek; Damian Egan; Fiona M. Doohan

    2009-01-01

    We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based

  15. Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

    PubMed Central

    Adamowicz, Michael S.; Stasulli, Dominique M.; Sobestanovich, Emily M.; Bille, Todd W.

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations. PMID:25549111

  16. Comparison of Rapid DNA Extraction Methods Applied to PCR Identification of Medicinal Mushroom Ganoderma spp

    Microsoft Academic Search

    Xuanwei Zhou; Qizhang Li; Jingya Zhao; Kexuan Tang; Juan Lin; Yizhou Yin

    2007-01-01

    Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm?broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high

  17. DNA Extraction From Processed Wood: A Case Study for the Identification of an Endangered

    E-print Network

    Protocols DNA Extraction From Processed Wood: A Case Study for the Identification of an Endangered to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered trop- ical timber species by using DNA sequencing technology. High-yield and high-quality DNA

  18. Stepping dsDNA through a solid-state nanopore one basepair at a time

    NASA Astrophysics Data System (ADS)

    Ho, Anthony; Comer, Jeffrey; Aksimentiev, Aleksei

    2011-03-01

    Solid-state nanopores hold great promise for single-molecule detection and manipulation, including low-cost, high-speed DNA sequencing. In a typical experiment, single molecules of DNA are driven through a nanopore by applying an electric potential difference across the membrane. As DNA passes through the pore, it modulates the ionic current, which potentially can be used to determine the DNA sequence. However, the typical rate of DNA transport in experiment is too high for detection of DNA sequences by ionic current measurement. It has been shown that it is possible to slow and weakly trap dsDNA in solid-state nanopores with diameters smaller than that of dsDNA [Nanotechnology 21:395501]. Using all-atom molecular dynamics simulations, we demonstrate that such pores can be used not only to trap but also to displace dsDNA in discrete steps using nanosecond-long pulses of electric field. Specifically, we have identified the pore geometry and pulse profiles that impel dsDNA by one basepair when the pulse is on and retain dsDNA in the same position when the pulse is off. Such nanopore traps may offer new means for manipulating single molecules in biophysics experiments.

  19. Optimized DNA extraction and metagenomic sequencing of airborne microbial communities.

    PubMed

    Jiang, Wenjun; Liang, Peng; Wang, Buying; Fang, Jianhuo; Lang, Jidong; Tian, Geng; Jiang, Jingkun; Zhu, Ting F

    2015-05-01

    Metagenomic sequencing has been widely used for the study of microbial communities from various environments such as soil, ocean, sediment and fresh water. Nonetheless, metagenomic sequencing of microbial communities in the air remains technically challenging, partly owing to the limited mass of collectable atmospheric particulate matter and the low biological content it contains. Here we present an optimized protocol for extracting up to tens of nanograms of airborne microbial genomic DNA from collected particulate matter. With an improved sequencing library preparation protocol, this quantity is sufficient for downstream applications, such as metagenomic sequencing for sampling various genes from the airborne microbial community. The described protocol takes ?12 h of bench time over 2-3 d, and it can be performed with standard molecular biology equipment in the laboratory. A modified version of this protocol may also be used for genomic DNA extraction from other environmental samples of limited mass or low biological content. PMID:25906115

  20. Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

    Microsoft Academic Search

    Hwan Young Lee; Myung Jin Park; Na Young Kim; Jeong Eun Sim; Woo Ick Yang; Kyoung-Jin Shin

    2010-01-01

    The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based

  1. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification.

    PubMed

    Pääbo, S

    1989-03-01

    Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics. PMID:2928314

  2. Extraction of Genomic DNA GTC Method Genomic DNA preps are useful for a number of applications including PCR and Southern

    E-print Network

    Extraction of Genomic DNA ­ GTC Method Genomic DNA preps are useful for a number of applications including PCR and Southern analysis. The following protocol for the GTC (guanidine thiocynate) method is a relatively faster protocol then the following CTAB Genomic DNA prep, but the yield of DNA can be variable

  3. A single-step competitive binding assay for mapping of single DNA molecules.

    PubMed

    Nyberg, Lena K; Persson, Fredrik; Berg, Johan; Bergström, Johanna; Fransson, Emelie; Olsson, Linnea; Persson, Moa; Stålnacke, Antti; Wigenius, Jens; Tegenfeldt, Jonas O; Westerlund, Fredrik

    2012-01-01

    Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence. PMID:22166208

  4. Stacking interactions in RNA and DNA: Roll-slide energy hyperspace for ten unique dinucleotide steps.

    PubMed

    Mukherjee, Sanchita; Kailasam, Senthilkumar; Bansal, Manju; Bhattacharyya, Dhananjay

    2015-03-01

    Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by ?B97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 134-147, 2015. PMID:25257334

  5. A Single-Step Method for Rapid Extraction of Total Lipids from Green Microalgae

    PubMed Central

    Axelsson, Martin; Gentili, Francesco

    2014-01-01

    Microalgae produce a wide range of lipid compounds of potential commercial interest. Total lipid extraction performed by conventional extraction methods, relying on the chloroform-methanol solvent system are too laborious and time consuming for screening large numbers of samples. In this study, three previous extraction methods devised by Folch et al. (1957), Bligh and Dyer (1959) and Selstam and Öquist (1985) were compared and a faster single-step procedure was developed for extraction of total lipids from green microalgae. In the single-step procedure, 8 ml of a 2?1 chloroform-methanol (v/v) mixture was added to fresh or frozen microalgal paste or pulverized dry algal biomass contained in a glass centrifuge tube. The biomass was manually suspended by vigorously shaking the tube for a few seconds and 2 ml of a 0.73% NaCl water solution was added. Phase separation was facilitated by 2 min of centrifugation at 350 g and the lower phase was recovered for analysis. An uncharacterized microalgal polyculture and the green microalgae Scenedesmus dimorphus, Selenastrum minutum, and Chlorella protothecoides were subjected to the different extraction methods and various techniques of biomass homogenization. The less labour intensive single-step procedure presented here allowed simultaneous recovery of total lipid extracts from multiple samples of green microalgae with quantitative yields and fatty acid profiles comparable to those of the previous methods. While the single-step procedure is highly correlated in lipid extractability (r2?=?0.985) to the previous method of Folch et al. (1957), it allowed at least five times higher sample throughput. PMID:24586930

  6. Immunoreaction-triggered DNA assembly for one-step sensitive ratiometric electrochemical biosensing of protein biomarker.

    PubMed

    Ren, Kewei; Wu, Jie; Yan, Feng; Zhang, Yue; Ju, Huangxian

    2015-04-15

    A sensitive ratiometric electrochemical readout was designed with an immunoreaction-triggered DNA assembly for one-step, fast and flexible assay of protein biomarker. The sensing interface was prepared by immobilizing a ferrocene (Fc)-labeled hairpin DNA on a gold electrode. In the presence of DNA2-antibody2 (Ab2) and methylene blue (MB)-labeled DNA1-Ab1 probes, the addition of target protein could induce the sandwich immunoreaction among two probes and the protein to trigger the hybridization of DNA1 and DNA2, which subsequently unfolded the hairpin DNA to form a three-arm DNA structure on the sensing interface. The DNA assembly caused the departure of Fc from the electrode and the approach of MB to the electrode, which led to the signal decrease and increase of Fc and MB respectively for ratiometric readout. Using prostate specific antigen (PSA) as a model target, the ratiometric electrochemical assay showed a linear detection range from 0.01 to 200ng/mL with a detection limit of 4.3pg/mL (the mean signal of blank measures+3?). By changing the affinity probe pairs this method could be easily expanded for other protein analytes, showing promising potential for point-of-care testing and extensive applications in bioanalysis. PMID:25460904

  7. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    PubMed

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-01

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine. PMID:21072429

  8. Akonni TruTip® and Qiagen® Methods for Extraction of Fetal Circulating DNA - Evaluation by Real-Time and Digital PCR

    PubMed Central

    Holmberg, Rebecca C.; Gindlesperger, Alissa; Stokes, Tinsley; Lopez, David; Hyman, Lynn; Freed, Michelle; Belgrader, Phil; Harvey, Jeanne; Li, Zheng

    2013-01-01

    Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods. PMID:23936545

  9. 2872 Biophysical Journal Volume 85 November 2003 28722883 DNA Basepair Step Deformability Inferred from Molecular

    E-print Network

    Langowski, Jörg

    . The force constants obtained are of similar magnitudes to those based on a crystallographic ensemble2872 Biophysical Journal Volume 85 November 2003 2872­2883 DNA Basepair Step Deformability Inferred elasticity, where the magnitude of deformation is proportional to the applied force (Hooke's law

  10. Early steps in the DNA base excision\\/single-strand interruption repair pathway in mammalian cells

    Microsoft Academic Search

    Muralidhar L Hegde; Tapas K Hazra; Sankar Mitra

    2008-01-01

    Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to

  11. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    Microsoft Academic Search

    Louise Vanysacker; Steven A. J. Declerck; Bart Hellemans; Luc De Meester; Ivo Vankelecom; Priscilla Declerck

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct\\u000a DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and\\u000a mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene.\\u000a Microbial community fingerprints were assessed by automated ribosomal

  12. Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.

    PubMed

    Heikal, Nahla; Nussenzveig, Roberto H; Agarwal, Archana M

    2014-09-01

    Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps). PMID:24897067

  13. Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.

    PubMed

    Lévy, Julien; Hancock, Joseph; Ravindran, Aravind; Gross, Dennis; Tamborindeguy, Cecilia; Pierson, Elizabeth

    2013-06-01

    This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids. PMID:23865212

  14. Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure.

    PubMed

    Castella, V; Dimo-Simonin, N; Brandt-Casadevall, C; Mangin, P

    2006-01-01

    The potential to recover genetic profiles from evidence samples has substantially increased since robust and sensitive amplification kits are commercially available. Nevertheless, even the best amplification kits cannot succeed when the extracted DNA is of poor quality. In this study we compared the efficiency of silica (QIAamp DNA Mini Kit), Chelex and Phenol-Chloroform (PC) based protocols to recover DNA from different categories of samples (blood and saliva on cotton swabs, muscles, cigarette butts, saliva on foods and epidermal cells on clothes). The efficiency of the QIAamp system was improved when samples were treated with QIAshredder homogenizing columns. Overall, conventional Chelex or PC protocols allowed to recover conclusive SGM Plus profiles for 61% of the samples considered in this study. Contrastingly, 82% of them were successfully genotyped after being treated with a combination of QIAshredder and QIAamp systems. Our results further suggested that the QIAshredder/QIAamp protocol was particularly helpful to analyze evidence samples with few DNA and/or that were collected on substrates containing PCR inhibitors. PMID:16326058

  15. Effectiveness of Salmon Carcass Tissue for Use in DNA Extraction and Amplification in Conservation Genetic Studies

    Microsoft Academic Search

    Jason Baumsteiger; Jacob L. Kerby

    2009-01-01

    A key concern in conservation genetic studies is obtaining viable DNA for analysis. In Pacific salmon Oncorhynchus spp., carcasses represent a feasible alternative for obtaining this tissue. However, the relative speed with which a salmon carcass decomposes can affect the quality of the extracted DNA. We extracted DNA from three different tissues (anal fin, operculum, and scales) obtained from carcasses

  16. Establishment and application of an efficient, economic, and rapid rice DNA extraction method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid, economic, and efficient method for DNA extraction from rice leaf, root and seed was developed, and the extracted DNA was used as a template to successfully amplify the rice blast resistance gene Pi-ta. Profiles of Pi-ta in 165 breeding lines detected by DNA markers were verified using diff...

  17. Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

    Microsoft Academic Search

    Julia R. de Lipthay; Christiane Enzinger; Kaare Johnsen; Jens Aamand; Søren J. Sørensen

    2004-01-01

    The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding–freezing–thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel

  18. A rapid non-destructive DNA extraction method for insects and other arthropods

    Microsoft Academic Search

    Mark A. Castalanelli; Dustin L. Severtson; Cameron J. Brumley; Andras Szito; Robert G. Foottit; Mike Grimm; Kylie Munyard; David M. Groth

    2010-01-01

    Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored

  19. Comparison of DNA extraction methods from muscle of canned tuna for species identification

    Microsoft Academic Search

    María José Chapela; Carmen G. Sotelo; Ricardo I. Pérez-Martín; Miguel Ángel Pardo; Begoña Pérez-Villareal; Patricia Gilardi; Juan Riese

    2007-01-01

    Four DNA extraction methods from canned tuna in different liquid media were compared. Wizard DNA Clean Up system with proteinase K previous digestion, Nucleospin (Clontech), GenomicPrep (Amersham Pharmacia Biotech) and the CTAB precipitation method were employed. DNA was extracted from four different canned tuna of the same tuna species, light tuna (Thunnus albacares): light tuna in brine, oil, vinegar and

  20. COMPARISON OF FOUR DNA EXTRACTION METHODS FROM INVASIVE FRESHWATER BIVALVE SPECIES (MOLLUSCA: BIVALVIA) IN ROMANIAN FAUNA

    Microsoft Academic Search

    OANA PAULA POPA; DUMITRU MURARIU; LUIS OVIDIU POPA

    2007-01-01

    In this paper we propose an evaluation of four DNA extraction methods in terms of DNA quantity, quality and success of the subsequent PCR amplifications of nuclear and mitochondrial loci. Individuals from the following freshwater invasive mussels in Romania were used to asses the efficiency of the DNA extraction methods: Dreissena polymorpha, Dreissena bugensis, Sinanodonta woodiana and Corbicula fluminea. While

  1. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction

    Microsoft Academic Search

    D P Jackson; F A Lewis; G R Taylor; A W Boylston; P Quirke

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase

  2. An improved method of extracting genomic DNA from preserved tissues of Capsicum annuum for PCR amplification

    Microsoft Academic Search

    Adebayo Liasu Ogunkanmi; Bola Oboh; Bukola Onifade; Adeniyi Adewale

    In this study we present a method for the extraction of genomic DNA from the different tissues of the Pepper (Capsicum annuum). A standard protocol of Dellaporta was reviewed and modified for DNA extraction from the preserved tissues of the Capsicum sample which was believed to contain high level of polysaccharides. The modified protocol employed yielded a high quality DNA

  3. DNA extraction methods for detecting genetically modified foods: A comparative study

    Microsoft Academic Search

    Rafaat M. Elsanhoty; Mohamed Fawzy Ramadan; Klaus Dieter Jany

    2011-01-01

    The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels

  4. Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

    PubMed

    Strotman, Lindsay N; Lin, Guangyun; Berry, Scott M; Johnson, Eric A; Beebe, David J

    2012-09-01

    Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 ?L of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods. PMID:22814365

  5. 'Direct PCR' optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction.

    PubMed

    Wong, Wing Hing; Tay, Ywee Chieh; Puniamoorthy, Jayanthi; Balke, Michael; Cranston, Peter S; Meier, Rudolf

    2014-11-01

    Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands. PMID:24816169

  6. A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing

    PubMed Central

    2014-01-01

    Background Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Methods Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment. PMID:25067909

  7. Comparative study of DNA extraction methods for soybean derived food products

    Microsoft Academic Search

    Isabel Mafra; Susana A. Silva; Elsa J. M. O. Moreira; Carla S. Ferreira da Silva; M. Beatriz; P. P. Oliveira

    2008-01-01

    The present work describes the comparison of four DNA extraction methods applied to a wide range of soybean derived food products. The methods included the commercial kits NucleoSpin and GeneSpin, the CTAB, and the Wizard methods. The protocols have been compared for their extraction efficiency, evaluated by the determination of yield and purity of DNA extracts, as well as amplifiability.

  8. DNA extracted from stained sputum smears can be used in the MTBDRplus assay.

    PubMed

    Dubois Cauwelaert, Natasha; Ramarokoto, Herimanana; Ravololonandriana, Pascaline; Richard, Vincent; Rasolofo, Voahangy

    2011-10-01

    We examined the feasibility of using DNA extracted from stained sputum smears for the detection of rifampin and isoniazid resistance with the commercial MTBDRplus assay from Hain Lifescience GmbH, Nehren, Germany. Overall sensitivity was initially low (70.0%) but increased to 96.7% when a multiplex PCR preamplification step was added. We then tested stored Mycobacterium tuberculosis-positive stained smears prepared from 297 patients' sputum samples. Species identification and drug susceptibility testing (DST) had been performed at the Institut Pasteur de Madagascar. Overall, the performance of the MTBDRplus assay applied to slide DNA was similar to that obtained in other studies with DNA extracted from clinical specimens. With the ready availability of stained smears in routine diagnostic laboratories and their easy transport and storage at room temperature, this approach should be useful for optimizing the treatment of multidrug-resistant tuberculosis and for conducting resistance surveys aimed at identifying hot-spot regions and breaking chains of transmission. PMID:21832013

  9. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

    Microsoft Academic Search

    L. Arlene Porteous; John L. Armstrong; Ramon J. Seidler; Lidia S. Watrud

    1994-01-01

    A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a

  10. Case study: using a nondestructive DNA extraction method to generate mtDNA sequences from historical chimpanzee specimens.

    PubMed

    Mohandesan, Elmira; Prost, Stefan; Hofreiter, Michael

    2012-01-01

    A major challenge for ancient DNA (aDNA) studies using museum specimens is that sampling procedures usually involve at least the partial destruction of each specimen used, such as the removal of skin, pieces of bone, or a tooth. Recently, a nondestructive DNA extraction method was developed for the extraction of amplifiable DNA fragments from museum specimens without appreciable damage to the specimen. Here, we examine the utility of this method by attempting DNA extractions from historic (older than 70 years) chimpanzee specimens. Using this method, we PCR-amplified part of the mitochondrial HVR-I region from 65% (56/86) of the specimens from which we attempted DNA extraction. However, we found a high incidence of multiple sequences in individual samples, suggesting substantial cross-contamination among samples, most likely originating from storage and handling in the museums. Consequently, reproducible sequences could be reconstructed from only 79% (44/56) of the successfully extracted samples, even after multiple extractions and amplifications. This resulted in an overall success rate of just over half (44/86 of samples, or 51% success), from which 39 distinct HVR-I haplotypes were recovered. We found a high incidence of C to T changes, arguing for both low concentrations of and substantial damage to the endogenous DNA. This chapter highlights both the potential and the limitations of nondestructive DNA extraction from museum specimens. PMID:22237528

  11. Comparison of DNA extraction methods for the PCR-single-strand-conformation polymorphism analysis of kefir

    Microsoft Academic Search

    Shu-Jun ChenQing; Qing Wang; Jian-Rong Han; Yu Wang; Zhi-Wei Li

    2011-01-01

    Experiments were performed to determine the influence of three DNA extraction methods (i.e. lysozyme, sonication and CTAB\\u000a methods) from kefir on the microbial diversity analysis by PCR-single strand conformation polymorphism (PCR-SSCP). The results\\u000a showed that the band of DNA extracted using CTAB was clearer than that using other methods. In addition, the yield and purity\\u000a of DNA extracted using CTAB

  12. Extraction of Genomic DNA CTAB-Lysozyme Method Isolation of genomic DNA is necessary for PCR and Southern analysis. The CTAB-

    E-print Network

    Extraction of Genomic DNA ­ CTAB-Lysozyme Method Isolation of genomic DNA is necessary for PCR and Southern analysis. The CTAB- Lysozyme method of genomic DNA extraction yields good quality DNA. Typically, the method yields good quality DNA that is suitable for Southern and PCR but not applications that require

  13. Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

    PubMed Central

    Wettig, Shawn; Slavcev, Roderick A.

    2014-01-01

    While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage ? pL promoter and regulated by the thermolabile ? CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ?90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

  14. Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

    PubMed

    Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

    2014-01-01

    While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage ? pL promoter and regulated by the thermolabile ? CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ? 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

  15. DNA Extraction by Isotachophoresis in a Microfluidic Channel

    SciTech Connect

    Stephenson, S J

    2011-08-10

    Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in an inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing electrolyte ions. Conversely, the trailing electrolyte ions have a slow electrophoretic mobility, so they lag behind the sample, thus trapping the species of interest between the LE and TE streams. In a typical isotachophoresis configuration, the electric field is applied in a direction parallel to the direction of flow. The species then form bands that stretch across the width of the channel. A major limitation of that approach is that only a finite amount of sample can be processed at once, and the sample must be processed in batches. For our purposes, a form of free-flow isotachophoresis is more convenient, where the DNA forms a band parallel to the edges of the channel. To achieve this, in our chip, the electric field is applied transversely. This creates a force perpendicular to the direction of flow, which causes the different ions to migrate across the flow direction. Because the mobility of the DNA is between the mobility of the leading and the trailing electrolyte, the DNA is focused in a tight band near the center of the channel. The stream of DNA can then be directed to a different output to produce a highly concentrated outlet stream without batch processing. One hurdle that must be overcome for successful ITP is isolating the electrochemical reactions that result from the application of high voltage for the actual process of isotachophoresis. The electrochemical reactions that occur around metal electrodes produce bubbles and pH changes that are detrimental to successful ITP. The design of the chips we use incorporates polyacrylamide gels to serve as electrodes along the central channel. For our design, the metal electrodes are located away from the chip, and high conductivity buffer streams carry the potential to the chip, functioning as a 'liquid electrode.' The stream then runs alongside a gel barrier. The gel electrode permits ion transfer while simultaneously isolating the separation chamber from any contaminants in the outer, 'liquid electrode' streams. The difference in potential from one side of the chip to the other creates an electric field. Thi

  16. A two-step strategy for constructing specifically self-subtracted cDNA libraries

    PubMed Central

    Laveder, Paolo; De Pittà, Cristiano; Toppo, Stefano; Valle, Giorgio; Lanfranchi, Gerolamo

    2002-01-01

    We have developed a new strategy for producing subtracted cDNA libraries that is optimized for connective and epithelial tissues, where a few exceptionally abundant (super-prevalent) RNA species account for a large fraction of the total mRNA mass. Our method consists of a two-step subtraction of the most abundant mRNAs: the first step involves a novel use of oligo-directed RNase H digestion to lower the concentration of tissue-specific, super-prevalent RNAs. In the second step, a highly specific subtraction is achieved through hybridization with probes from a 3?-end ESTs collection. By applying this technique in skeletal muscle, we have constructed subtracted cDNA libraries that are effectively enriched for genes expressed at low levels. We further report on frequent premature termination of transcription in human muscle mitochondria and discuss the importance of this phenomenon in designing subtractive approaches. The tissue-specific collections of cDNA clones generated by our method are particularly well suited for expression profiling. PMID:11972353

  17. Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

    Microsoft Academic Search

    Nobuyuki Ikeda; Nonnatus S. Bautista; Tetsuya Yamada; Osamu Kamijima; Takashige Ishii

    2001-01-01

    We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube,\\u000a one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions.\\u000a The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals

  18. A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Takata, Tatsuya; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2010-06-01

    We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

  19. Extraction of genomic DNA from yeasts for PCR-based applications.

    PubMed

    Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

    2011-05-01

    We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ? 3500 bp. PMID:21548894

  20. Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension)

    E-print Network

    Beebe, David J.

    and hazardous chemicals, however the advent of DNA solid phase extraction methods (SPE) drastically changedFacile and rapid DNA extraction and purification from food matrices using IFAST (immiscible Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample

  1. HELICASE DEPENDENT AMPLICATION transfer the DNA

    E-print Network

    STEP 1 STEP 2 STEP 3 HELICASE DEPENDENT AMPLICATION HELICASE DNA POLYMERASE transfer the DNA SAMPLES INto a microfluidic CHIP ALONG WITH The Amplification Master mix Reagents. DNA amplification OF TOE WARMERS. use "snap" to extract your dNA or RNA from the HUman Sample. Put it all together

  2. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

    PubMed Central

    Moncada, Ximena; Payacán, Claudia; Arriaza, Francisco; Lobos, Sergio; Seelenfreund, Daniela; Seelenfreund, Andrea

    2013-01-01

    Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials. PMID:23437166

  3. Estimation of the uncertainties of extraction and clean-up steps in pesticide residue analysis of plant commodities.

    PubMed

    Omeroglu, P Yolci; Ambrus, A; Boyacioglu, D

    2013-01-01

    Extraction and clean-up constitute important steps in pesticide residue analysis. For the correct interpretation of analytical results, uncertainties of extraction and clean-up steps should be taken into account when the combined uncertainty of the analytical result is estimated. In the scope of this study, uncertainties of extraction and clean-up steps were investigated by spiking (14)C-labelled chlorpyrifos to analytical portions of tomato, orange, apple, green bean, cucumber, jackfruit, papaya and starfruit. After each step, replicate measurements were carried out with a liquid scintillation counter. Uncertainties in extraction and clean-up steps were estimated separately for every matrix and method combination by using within-laboratory reproducibility standard deviation and were characterised with the CV of recoveries. It was observed that the uncertainty of the ethyl acetate extraction step varied between 0.8% and 5.9%. The relative standard uncertainty of the clean-up step with dispersive SPE used in the method known as QuEChERS was estimated to be around 1.5% for tomato, apple and green beans. The highest variation of 4.8% was observed in cucumber. The uncertainty of the clean-up step with gel permeation chromatography ranged between 5.3% and 13.1%, and it was relatively higher than that obtained with the dispersive SPE method. PMID:23216411

  4. Commercial DNA extraction kits impact observed microbial community composition in permafrost samples.

    PubMed

    Vishnivetskaya, Tatiana A; Layton, Alice C; Lau, Maggie C Y; Chauhan, Archana; Cheng, Karen R; Meyers, Arthur J; Murphy, Jasity R; Rogers, Alexandra W; Saarunya, Geetha S; Williams, Daniel E; Pfiffner, Susan M; Biggerstaff, John P; Stackhouse, Brandon T; Phelps, Tommy J; Whyte, Lyle; Sayler, Gary S; Onstott, Tullis C

    2014-01-01

    The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA(®) SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil(®) (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed ?- and ?-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits. PMID:24102625

  5. Increasing DNA extraction yield from saliva stains with a modified Chelex method

    Microsoft Academic Search

    David Sweet; Miguel Lorente; Aurora Valenzuela; José A. Lorente; J. Carlos Alvarez

    1996-01-01

    Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results

  6. Kemp Lab Ancient DNA Extraction Protocol-"Old" Method Updated by Brian M. Kemp, September 2012

    E-print Network

    Kemp, Brian M.

    Kemp Lab Ancient DNA Extraction Protocol- "Old" Method Updated by Brian M. Kemp, September 2012 Members of the Kemp Lab have come to affectionately call this protocol the "old" method, in contrast to the "new" method as described in the "Kemp Lab Ancient DNA Extraction Protocol- `New' Method" document

  7. RECOVERY OF BULK DNA FROM SOIL USING A RAPID, SMALL-SCALE EXTRACTION METHOD

    EPA Science Inventory

    We describe an extraction method that yields restrictable 20-25 kb DNA from one gram of soil. ells are lysed directly in the soil. he crude DNA extract is separated from contaminating humic compounds, concentrated, and purified by CsCl gradient centrifugation and the commercial p...

  8. 96-well Format DNA Extraction Protocol for Freeze-dried Maize Seedling Leaves

    E-print Network

    Wurtele, Eve Syrkin

    , 1985) it is possible to extract DNA from 8 x 96 maize samples/day. Additional modification (provided1 96-well Format DNA Extraction Protocol for Freeze-dried Maize Seedling Leaves (Last revised. Marna Yandeau and Dave Skibbe. Using this protocol (a modification of one provided by Rogers and Bendich

  9. Influence of Extraction Protocol on Physical Properties of Parvovirus B19 DNA ?

    PubMed Central

    Matz, Bertfried; Kupfer, Bernd; Kreil, Thomas R.; Eis-Hübinger, Anna Maria

    2010-01-01

    While establishing a quantification standard for parvovirus B19 diagnostics, we extracted viral DNA from a high-titered human plasma by using three commercial kits. Despite similar viral DNA yields being obtained, striking differences in electrophoretic mobilities of the extracted nucleic acids were observed. PMID:20943865

  10. A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

    EPA Science Inventory

    Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

  11. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps

    PubMed Central

    Park, Jeehae; Myong, Sua; Niedziela-Majka, Anita; Yu, Jin; Lohman, Timothy M.; Ha, Taekjip

    2010-01-01

    Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA bound proteins, and basic properties like the elementary step size remain controversial. Using single molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single stranded loop. Static disorder limited previous ensemble studies of PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations suggesting a mode of action for eliminating potentially deleterious recombination intermediates. PMID:20723756

  12. EFFECTIVE METHOD TO EXTRACT DNA FROM ENVIRONMENTAL SAMPLES FOR POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA FINGERPRINT ANALYSIS

    EPA Science Inventory

    A rapid direct-extraction method was used to obtain DNA from environmental soil samples. eat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. he DNA was purified by agarose gel electrophoresis, amplified with 16S based primers by use of the polymerase chain rea...

  13. Direct DNA Extraction for PCR-Mediated Assays of Soil Organisms

    Microsoft Academic Search

    TATIANA VOLOSSIOUK; E. JANE ROBB; ANDROSS N. NAZAR

    1995-01-01

    By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and

  14. Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon

    Microsoft Academic Search

    Lu Zheng; Naiyun Gao; Yang Deng

    2012-01-01

    It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes

  15. Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon

    Microsoft Academic Search

    Lu Zheng; Naiyun Gao; Yang Deng

    2011-01-01

    It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes

  16. Comparison of DNA extraction methods for pathogenic Yersinia enterocolitica detection from meat food by nested PCR

    Microsoft Academic Search

    Cecilia S. M. Lucero Estrada; Lidia del Carmen Velázquez; Silvia Di Genaro; Ana María Stefanini de Guzmán

    2007-01-01

    The objective of this work was to compare three different methods of DNA extraction from meat food, and to determine whether these methods removed inhibitors of nested PCR for pathogenic Yersinia enterocolitica detection. The amplification of the yadA gene from the DNA obtained from a pure Y. enterocolitica culture could be carried out with all the protocols. DNA amplification from

  17. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Microsoft Academic Search

    Tae-Jin Kang; Moon-Sik Yang

    2004-01-01

    BACKGROUND: DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed.

  18. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost

    E-print Network

    Michel Jr., Frederick C.

    Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost complicate the isolation of PCR- amplifiable DNA from compost and other organic-rich samples. In this study from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel

  19. DNA is a co-factor for its own replication in Xenopus egg extracts

    E-print Network

    DNA is a co-factor for its own replication in Xenopus egg extracts Ronald Lebofsky1 , Antoine M a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA repli- cation in this system is highly sensitive to plasmid concentration, being undetectable

  20. STRs typing of DNA extracted from cigarette butts soaked in flammable liquids for several weeks

    Microsoft Academic Search

    M. Pizzamiglio; A. Marino; G. Maugeri; L. Garofano

    2006-01-01

    This paper refers to a case of arson, in which we analyzed three cigarette butts, apparently smoked, collected from a crime scene. They were soaked in a petroleum blend and used to ignite the fire. DNA extraction was carried out using QIAamp 96 DNA Swab BioRobot kit procedure. The amount of human DNA recovered was then quantified by slot-blot hybridization

  1. DNA Extraction 5th Grade Physical Science Standard 1.1

    E-print Network

    DNA Extraction 5th Grade Physical Science Standard 1.1 Concepts and Skills: Mixtures of matter can don't mix. 5. Gently pull DNA strands from the bottom solution into the isopropanol with a stir rod. DNA should precipitate into fine white strands that look like cotton candy. What is happening? Mashing

  2. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    PubMed

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples. PMID:25143727

  3. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

    PubMed Central

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

  4. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

    PubMed

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

  5. Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

    PubMed Central

    2014-01-01

    Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96%?v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30?days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue. PMID:24761774

  6. DNA extraction from bloodstains in respect to age and stained substrate.

    PubMed

    Prinz, M; Staak, M; Berghaus, G

    1989-01-01

    The amount and the quality of DNA that could be extracted from bloodstains equivalent to 200 microliters blood were examined after 3, 6, 9, 12, 15, 18 and 21 days of storage under dry or humid conditions at room temperature. DNA was also extracted from two days old 200 microliters bloodstains on different stain carriers. The amount of DNA that could be extracted showed a dependency on all the parameters that were examined. On carriers with a rough surface structure, where the blood can soak in, the resolving of leukocytes was impaired and the DNA recovery rate was low, e.g. carpeting and suede. The DNA yield was higher for substrates with a smooth surface, e.g. paper, glass and smooth leather. The aging experiments revealed that for the stains stored under dry conditions, there was no decrease in the DNA yield for stains on glass, while the amount of DNA that could be extracted became less for older stains on cotton. Under humid conditions the DNA yield was high and did not decrease for both glass and cotton fabric. The quality of the DNA was not impaired by aging or storage conditions, but for several stain carriers the DNA was contaminated by chemical substances which inhibited restriction enzyme digestion and successful DNA-typing. PMID:2535364

  7. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  8. Detection of BK virus in urine by polymerase chain reaction: a comparison of DNA extraction methods

    Microsoft Academic Search

    A Behzadbehbahani; P. E Klapper; P. J Vallely; G. M Cleator

    1997-01-01

    Using specimens spiked with BK virus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from urine samples. It was found that PCR inhibition could be completely overcome by extracting samples with 30% polyethylene glycol (PEG) in 3 M sodium chloride, and partially overcome by extracting samples with guanidine thiocyanate in the presence

  9. Effect of DNA extraction and sample preservation method on rumen bacterial population.

    PubMed

    Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kope?ný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

    2014-10-01

    The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. PMID:24125910

  10. One of the key characteristics of ancient DNA, low copy number, may be a product of its extraction

    E-print Network

    Kemp, Brian M.

    developed synthesized "standards" to measure the efficiency of some common DNA extraction methods to a given extraction method (i.e. "copies in") versus quantity of DNA retained (i.e. "copies out"). All methods performed poorly in retaining short segments of DNA, giving low copy number results even when pre-extraction

  11. Efficiency of genomic DNA extraction dependent on the size of magnetic nanoclusters

    NASA Astrophysics Data System (ADS)

    Cho, Hyun Ah; Hyun Min, Ji; Hua Wu, Jun; Woo Jang, Jin; Lim, Chae-Seung; Keun Kim, Young

    2014-05-01

    We report the efficiency of genomic DNA extraction as a function of particle size and quantity. For DNA extraction, we synthesized magnetic nanoclusters of various sizes and coated the surface of these magnetic nanoclusters with meso-2,3-dimercaptosuccinic acid. We showed that the nanoclusters had a tight particle size distribution and high crystallinity. Furthermore, we observed that the three types of magnetic nanoclusters studied exhibited ferrimagnetic behavior and that larger nanoclusters showed larger saturation magnetization values. The resultant efficiency of DNA extraction is inversely proportional to particle size in the range of nanoclusters tested, due to the fact that the surface-to-volume ratio decreases as particle size increases.

  12. Extraction of total DNA and optimization of the RAPD reaction system in Dioscorea opposita Thunb.

    PubMed

    Wen, G Q; Li, J; Liu, X H; Zhang, Y S; Wen, S S

    2014-01-01

    Dioscorea opposita Thunb. has been used as health food and herbal medicinal ingredients in traditional Chinese medicine. In this study, the total DNA of D. opposita Thunb. was extracted using an improved cetyltrimethylammonium bromide (CTAB) method, and the extracted DNA was further used for random amplified polymorphic DNA (RAPD) reaction system by design of the L16 (4(4)) orthogonal diagram. The results showed that the improved CTAB method can be used to isolate high-quality and high-concentration DNA, and the optimized protocol can overcome the instability of RAPD reaction system. The knowledge stated here can be used to study the genetic diversity of D. opposita Thunb. PMID:24634232

  13. DNA encoding based feature extraction for biometric watermarking

    Microsoft Academic Search

    Meenakshi S Arya; Nikita Jain; Jai Sisodia; Nukul Sehgal

    2011-01-01

    The aim of this paper is to secure the digital code of a watermark (which is an offline handwritten signature) by using the characteristics of DNA. The watermarked image is embedded into the image as binary information and further encrypted as DNA sequences and these DNA sequences (after being grouped as tri - nucleotide sequences called codons) then attached to

  14. Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

    Microsoft Academic Search

    William R. Hudlow; Martin R. Buoncristiani

    We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic\\/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The

  15. Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection

    PubMed Central

    Romano, Christine A.; Sontz, Pamela A.; Barton, Jacqueline K.

    2011-01-01

    Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30, Y55, Y75 and Y82 help mediate charge transport between DNA and the [4Fe-4S] cluster. Based on circular dichroism studies to measure protein stability, mutations at residues W178 and Y185 are found to destabilize the protein; these residues may function to protect the [4Fe-4S] cluster. Atomic force microscopy studies furthermore reveal a correlation in the ability of mutants to carry out protein/DNA CT and their ability to relocalize onto DNA strands containing a single base mismatch; EndoIII mutants that are defective in carrying out DNA/protein CT do not redistribute onto mismatch-containing strands, consistent with our model. These results demonstrate a link between the ability of the repair protein to carry out DNA CT and its ability to relocalize near lesions, thus pointing to DNA CT as a key first step in the detection of base damage in the genome. PMID:21651304

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

    PubMed

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

    2014-06-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations. PMID:24838067

  17. Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET.

    PubMed

    Chen, Hunter H; Ho, Yi-Ping; Jiang, Xuan; Mao, Hai-Quan; Wang, Tza-Huei; Leong, Kam W

    2009-04-01

    Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously. PMID:20161048

  18. Repeat silica extraction: a simple technique for the removal of PCR inhibitors from DNA extracts

    E-print Network

    Kemp, Brian M.

    to conclude that negative PCR amplification is the result of the absence of preserved DNA. Ã? 2006 Elsevier Ltd], postmortem DNA dam- age [2], and DNA preservation [3]. One less explored topic in aDNA research of food and environ- mental samples [4,24e27]. The presence of PCR inhibitors is often, but not always, in

  19. Qualification Study of Two Genomic DNA Extraction Methods in Different Clinical Samples

    PubMed Central

    Javadi, Alireza; Shamaei, Masoud; Pourabdollah, Mihan; Dorudinia, Atosa; Seyedmehdi, Seyed Mohammad; Karimi, Shirin

    2014-01-01

    Introduction The purity of genomic DNA (gDNA) extracted from different clinical specimens optimizes sensitivity of polymerase chain reaction (PCR) assays. This study attempted to compare two different DNA extraction techniques namely salting-out and classic phenol-chloroform. Materials and Methods Qualification of two different DNA extraction techniques for 634 clinical specimens highly suspected of having mycobacterial infection was performed. Genomic DNA was extracted from 330 clinical samples using phenol-chloroform and 304 by non-toxic salting-out. Qualification of obtained gDNA was done through amplification of internal controls, ?-actin and ?-globin. Results ?-actin-positive was detected in 279/330 (84%) and 272/304 (89%) samples by phenol-chloroform technique and salting-out, respectively. PCR inhibitor was found for the gDNA of 13/304 (4%) patient samples were negative by ?-actin and ?-globin tests via salting-out technique in comparison with gDNAs from 27/330 (8.5%) samples extracted by phenol-chloroform procedure. No statistically significant difference was found between phenol-chloroform technique and salting-out for 385 sputum, 29 bronchoalveolar lavage (BAL), 105 gastric washing, and 38 body fluid (P=0.04) samples. This illustrates that both techniques have the same quality for extracting gDNA. Conclusion This study discloses salting-out as a non-toxic DNA extraction procedure with a superior time-efficiency and cost-effectiveness in comparison with phenol-chloroform and it can be routinely used in resource-limited laboratory settings.

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

    PubMed

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

    2014-09-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples. PMID:25197027

  1. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species

    Microsoft Academic Search

    Muhammad A. Lodhi; Guang-Ning Ye; Norman F. Weeden; Bruce I. Reisch

    1994-01-01

    A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure\\u000a modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method\\u000a has also been used successfully

  2. Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods

    Microsoft Academic Search

    G. Amagliani; C. Giammarini; E. Omiccioli; G. Brandi; M. Magnani

    2007-01-01

    The aim of our work was to evaluate a new commercial test kit for the detection of Listeria monocytogenes by PCR, using different DNA extraction methods. Food samples (pork sausage and “mozzarella” cheese) were spiked with known concentrations of L. monocytogenes and culture-enriched for 24h. DNA extracted using three commercial kits and two standard methods, was amplified in species-specific PCR

  3. DNA extraction method affects microbial community profiles from soils and sediment

    Microsoft Academic Search

    Cora Carrigg; Olivia Rice; Siobhán Kavanagh; Gavin Collins; Vincent O’Flaherty

    2007-01-01

    To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community\\u000a composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we\\u000a compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by\\u000a polymerase chain reaction of purified DNA extracts, demonstrated that the

  4. A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material

    Microsoft Academic Search

    Beverley C. Millar; Xu Jiru; John E. Moore; John A. P. Earle

    2000-01-01

    This study investigated the various commercially available kits and ‘in-house’ methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads\\/sonication and wash\\/alkali\\/heat lysis. The results indicated that

  5. DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts

    Microsoft Academic Search

    Domenico Bosco; Simona Palermo; Giovanna Mason; Rosemarie Tedeschi; Cristina Marzachì; Guido Boccardo

    2002-01-01

    DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and\\/or field-collected leafhoppers\\u000a and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved\\u000a by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the\\u000a 16\\/23S ribosomal primer pairs used and the specific assay and cycling

  6. Xenopus laevis Ctc1-Stn1-Ten1 (xCST) Protein Complex Is Involved in Priming DNA Synthesis on Single-stranded DNA Template in Xenopus Egg Extract*

    PubMed Central

    Nakaoka, Hidenori; Nishiyama, Atsuya; Saito, Motoki; Ishikawa, Fuyuki

    2012-01-01

    The Ctc1-Stn1-Ten1 (CST) complex is an RPA (replication protein A)-like protein complex that binds to single-stranded (ss) DNA. It localizes at telomeres and is involved in telomere end protection in mammals and plants. It is also known to stimulate DNA polymerase ?-primase in vitro. However, it is not known how CST accomplishes these functions in vivo. Here, we report the identification and characterization of Xenopus laevis CST complex (xCST). xCST showed ssDNA binding activity with moderate preference for G (guanine)-rich sequences. xStn1-immunodepleted Xenopus egg extracts supported chromosomal DNA replication in in vitro reconstituted sperm nuclei, suggesting that xCST is not a general replication factor. However, the immunodepletion or neutralization of xStn1 compromised DNA synthesis on ssDNA template. Because primed ssDNA template was replicated in xStn1-immunodepleted extracts as efficiently as in control ones, we conclude that xCST is involved in the priming step on ssDNA template. These results are consistent with the current model that CST is involved in telomeric C-strand synthesis through the regulation of DNA polymerase ?-primase. PMID:22086929

  7. Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

    PubMed Central

    Almouzni, G; Méchali, M

    1988-01-01

    A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork. Images PMID:3396538

  8. Single-molecule analysis of DNA replication in Xenopus egg extracts Hasan Yardimci a

    E-print Network

    Single-molecule analysis of DNA replication in Xenopus egg extracts Hasan Yardimci a , Anna B: Available online 6 April 2012 Communicated by Marcel Mechali Keywords: Single-molecule DNA replication Fluorescence microscopy a b s t r a c t The recent advent in single-molecule imaging and manipulation methods

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

    PubMed Central

    Khare, Parul; Raj, Vineet; Chandra, Shaleen; Agarwal, Suraksha

    2014-01-01

    Saliva has long been known for its diagnostic value in several diseases. It also has a potential to be used in forensic science. Objective: The objective of this study is to compare the quantity and quality of DNA samples extracted from saliva with those extracted from blood in order to assess the feasibility of extracting sufficient DNA from saliva for its possible use in forensic identification. Materials and Methods: Blood and saliva samples were collected from 20 volunteers and DNA extraction was performed through Phenol Chloroform technique. The quantity and quality of isolated DNA was analyzed by spectrophotometery and the samples were then used to amplify short tandem repeat (STR) F13 using the polymerase chain reaction. Results: Mean quantity of DNA obtained in saliva was 48.4 ± 8.2 ?g/ml and in blood was 142.5 ± 45.9 ?g/ml. Purity of DNA obtained as assessed by the ratio of optical density 260/280, was found to be optimal in 45% salivary samples while remaining showed minor contamination. Despite this positive F13 STR amplification was achieved in 75% of salivary DNA samples. Conclusion: Results of this study showed that saliva may prove to be a useful source of DNA for forensic purpose. PMID:25125913

  10. Extraction platform evaluations: A comparison of Automate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems

    Microsoft Academic Search

    Carey P. Davis; Jonathan L. King; Bruce Budowle; Arthur J. Eisenberg; Meredith A. Turnbough

    The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life

  11. Genomic DNA extraction from small amounts of serum to be used for  1-antitrypsin genotype analysis

    Microsoft Academic Search

    S. Andolfatto; F. Namour; A. L. Garnier; F. Chabot; J. L. Gueant; I. Aimone-Gastin

    2003-01-01

    If laboratory diagnosis of a1-antitrypsin (a1-AT) deficiency is usually based on its phenotype identification by isoelectric focusing, a1-antiprotease inhibitor (Pi)S and PiZ genotypes can also be determined by deoxyribonucleic acid (DNA)-based methods. Recently, several methods have been described for preparing genomic DNA from serum. The aim of the current study was to determine the Pi allele from serum extracted DNA

  12. PCR-based typing of DNA extracted from cigarette butts

    Microsoft Academic Search

    M. N. Hochmeister; R. Dirnhofer; U. V. Borer; B. Budowle; J. Jung; C. T. Comey

    1991-01-01

    Summary Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime

  13. Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.*

    PubMed Central

    Azmat, Muhammad Abubakkar; Khan, Iqrar Ahmad; Cheema, Hafiza Masooma Naseer; Rajwana, Ishtiaq Ahmad; Khan, Ahmad Sattar; Khan, Asif Ali

    2012-01-01

    Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and ?-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950?1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems. PMID:22467363

  14. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p?extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation. PMID:25829616

  15. A DNA trapping and extraction microchip using periodically crossed electrophoresis in a micropillar array

    Microsoft Academic Search

    Soyeon Yi; Kyoung-Sun Seo; Young-Ho Cho

    2005-01-01

    This paper presents an integrated deoxyribose nucleic acid (DNA) trapping and extraction microchip based on the electrophoresis using periodically crossed electric fields in the micropillar array. The extraction microchip, integrated with a micropillar array, microchannels, nano-gap entropic barriers, loading and unloading windows, has been fabricated by a 3-mask microfabrication process. Using the electric field crossed at 120°, the microchip is

  16. Extraction of high quality DNA from biological materials and calcified tissues

    Microsoft Academic Search

    James Stray; Allison Holt; Maxim Brevnov; Lisa M. Calandro; Manohar R. Furtado; Jaiprakash G. Shewale

    2009-01-01

    Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and

  17. High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods

    Microsoft Academic Search

    Gracia M. Martín-Núñez; Juan M. Gómez-Zumaquero; Federico Soriguer; Sonsoles Morcillo

    BackgroundHigh resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations.

  18. Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples

    Microsoft Academic Search

    Christopher A. Whittier; Arun K. Dhar; Chip Stem; Jane Goodall; Acacia Alcivar-Warren

    1999-01-01

    Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v\\/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal

  19. Cloud point extraction as a preconcentration step prior to capillary electrophoresis.

    PubMed

    Carabias-Martínez, R; Rodríguez-Gonzalo, E; Domínguez-Alvarez, J; Hernández-Méndez, J

    1999-07-01

    Cloud point extraction was applied as a preconcentration step prior to capillary electrophoresis. The behavior of a surfactant-rich micellar phase injected into a capillary electrophoresis system was studied using different separation modes:? micellar electrokinetic capillary chromatography and capillary zone electrophoresis (CZE). A problem that appeared on introducing a surfactant-rich phase into a bare fused silica capillary was that the surfactant was adsorbed onto the wall of the capillary, leading to a marked loss of efficiency and reproducibility both in the migration times and in the areas of the electrophoretic peaks. The use of cetyltrimethylammonium bromide dynamically coated capillaries afforded reproducible results, although the half-life of the capillary was short. The most satisfactory results were obtained by using nonaqueous media in the CZE mode, thus avoiding surfactant adsorption. Other parameters related to the composition of the injection medium were also studied to optimize the electrophoretic behavior of the analytes and the sensitivity of the determination. The optimized procedure was applied to the determination of triazines in tap and river water samples. PMID:21662790

  20. Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract.

    PubMed Central

    Weinfeld, M; Drake, A F; Saunders, J K; Paterson, M C

    1985-01-01

    The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract. Images PMID:3903661

  1. A three-step molecular protocol employing DNA obtained from dried blood spots for neonatal screening for 45,X Turner syndrome.

    PubMed

    Rocha, Mylene Neves; Melo, Murilo Rezende; Longui, Carlos Alberto; de Oliveira, Daniela Vilariço Alves; Figueiredo, Carolina Costa; Pacchi, Paulo Roberto

    2005-01-01

    Turner syndrome (TS) is one of the most common human chromosomal abnormalities; it is characterized by the presence of one normal X chromosome and the complete or partial loss of the second X chromosome. The early recognition of TS patients allows for adequate therapy for short stature and pubertal sex steroid substitution. We developed a cost-effective molecular diagnostic tool that can be used to identify 45,X TS patients from dried blood spots, for possible use in neonatal screening for TS. We used a three-step method for 45,X TS detection: i) DNA extraction from dried blood spot samples, ii) pre-PCR HpaII digestion (methylation-sensitive enzyme) and iii) GeneScan analysis of selected cases. DAX-1 gene amplification was used to recognize DNA integrity, and the androgen receptor gene (Xq11-12), which is both a highly polymorphic and methylated gene, was used to determine the number of X chromosome alleles. Using this three-step diagnostic procedure, we detected apparent TS in 1/304 (0.33%) samples; such individuals should be submitted to clinical examination and karyotype confirmation. The three-step 45,X TS neonatal screening protocol is a simple, reliable, fast (under 30 h) and cost-effective diagnostic tool, useful for the neonatal detection of TS. PMID:16475121

  2. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  3. Effect of thyme (T. vulgaris) extracts on fattening performance, some blood parameters, oxidative stress and DNA damage in Japanese quails

    Microsoft Academic Search

    T. Sengül; S. Yurtseven; M. Cetin; A. Kocyigit; B. Sögüt

    The study was conducted to determine the effects of supplemented thyme oil extract and thyme water extract, the water soluble fraction of thyme extract, on fattening performance, blood parameters, oxidative stress and DNA damage in Japanese quails. Two hundred sixteen chicks were divided into four groups: control (no antibiotic or thyme extracts (I), fl avomycin (II), thyme oil extract (III)

  4. Repair of a synthetic abasic site in DNA in a Xenopus laevis oocyte extract.

    PubMed Central

    Matsumoto, Y; Bogenhagen, D F

    1989-01-01

    Covalently closed circular DNA containing a synthetic analog of an abasic site at a unique position was used as a substrate to study DNA repair. Incubation of this DNA in Xenopus laevis oocyte extracts resulted in rapid cleavage of the DNA at the abasic site by a class II apurinic-apyrimidinic endonuclease, followed by complete repair within 40 min. Nicked circular DNAs persisted for several minutes before repair by an ATP-dependent DNA synthesis reaction. The repair-related DNA synthesis was localized within 3 or 4 nucleotides surrounding the abasic site. These results are consistent with the short-patch repair reported for DNA damage at heterogeneous sites in human cells (J. D. Regan and R. B. Setlow, Cancer Res. 34:3318-3325, 1974). Images PMID:2779565

  5. Comparison of three DNA extraction kits to establish maximum yield and quality of coral-associated microbial DNA

    USGS Publications Warehouse

    Baker, Erin J.; Kellogg, Christina A.

    2014-01-01

    Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc., have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.

  6. Plant DNA detection from grasshopper guts: A step-by-step protocol, from tissue preparation to obtaining plant DNA sequences1

    PubMed Central

    Avanesyan, Alina

    2014-01-01

    • Premise of the study: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. • Methods and Results: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA) gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. • Conclusions: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant “movement” during food consumption, to detecting plant–insect interactions. PMID:25202604

  7. Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

    Microsoft Academic Search

    Tina Mygind; Lars Østergaard; Svend Birkelund; Jes S Lindholt; Gunna Christiansen

    2003-01-01

    BACKGROUND: To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue. RESULTS: The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with

  8. [Extraction and purification method of rice DNA from rice powder containing Konjak flour].

    PubMed

    Minematsu, Kazuhiko; Nakamura, Kosuke; Akiyama, Hiroshi; Harikai, Naoki; Nakajima, Osamu; Kitta, Kazumi; Teshima, Reiko; Iizuka, Tayoshi

    2010-01-01

    Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR. PMID:21071909

  9. Utility of arsenic-treated bird skins for DNA extraction

    PubMed Central

    2011-01-01

    Background Natural history museums receive a rapidly growing number of requests for tissue samples from preserved specimens for DNA-based studies. Traditionally, dried vertebrate specimens were treated with arsenic because of its toxicity and insect-repellent effect. Arsenic has negative effects on in vivo DNA repair enzymes and consequently may inhibit PCR performance. In bird collections, foot pad samples are often requested since the feet were not regularly treated with arsenic and because they are assumed to provide substantial amounts of DNA. However, the actual influence of arsenic on DNA analyses has never been tested. Findings PCR success of both foot pad and body skin samples was significantly lower in arsenic-treated samples. In general, foot pads performed better than body skin samples. Moreover, PCR success depends on collection date in which younger samples yielded better results. While the addition of arsenic solution to the PCR mixture had a clear negative effect on PCR performance after the threshold of 5.4 ?g/?l, such high doses of arsenic are highly unlikely to occur in dried zoological specimens. Conclusions While lower PCR success in older samples might be due to age effects and/or DNA damage through arsenic treatment, our results show no inhibiting effect on DNA polymerase. We assume that DNA degradation proceeds more rapidly in thin tissue layers with low cell numbers that are susceptible to external abiotic influences. In contrast, in thicker parts of a specimen, such as foot pads, the outermost horny skin may act as an additional barrier. Since foot pads often performed better than body skin samples, the intention to preserve morphologically important structures of a specimen still conflicts with the aim to obtain optimal PCR success. Thus, body skin samples from recently collected specimens should be considered as alternative sources of DNA. PMID:21676254

  10. Dichlorvos exposure impedes extraction and amplification of DNA from insects in museum collections

    PubMed Central

    2010-01-01

    Background The insecticides dichlorvos, paradichlorobenzene and naphthalene have been commonly used to eradicate pest insects from natural history collections. However, it is not known how these chemicals affect the DNA of the specimens in the collections. We thus tested the effect of dichlorvos, paradichlorobenzene and naphthalene on DNA of insects (Musca domestica) by extracting and amplifying DNA from specimens exposed to insecticides in two different concentrations over increasing time intervals. Results The results clearly show that dichlorvos impedes both extraction and amplification of mitochondrial and nuclear DNA after relatively short time, whereas paradichlorobenzene and naphthalene do not. Conclusion Collections treated with paradichlorobenzene and naphthalene, are better preserved concerning DNA, than those treated with dichlorvos. Non toxic pest control methods should, however, be preferred due to physical damage of specimens and putative health risks by chemicals. PMID:20148102

  11. Nondestructive methods for recovery of biological material from human teeth for DNA extraction.

    PubMed

    Hervella, Montserrat; Iñiguez, Maitane G; Izagirre, Neskuts; Anta, Alberto; de-la-Rúa, Concepción

    2015-01-01

    The extraction of DNA from human skeletal remains applied to forensic, and evolutionary studies do not exclude risks, which are to be evaluated when working with unique specimens that could be damaged or even destroyed. In the present study were evaluated several nondestructive methods for recovering DNA instead of the most currently used pulverization method. Three different procedures to access inside the dental pieces (occlusal perforation, cervical perforation, and cervical cut) have been compared with the aim of recovering as many cell remains as possible to carry out a DNA extraction. Given the DNA quantitation results, a method was proposed that consists of a cervical cut to facilitate the access to the pulp cavity and a subsequent filing of the root canals down to the apex of the dental root. This methodology allows the recovery of both mitochondrial and nuclear DNA, with the minimum deterioration for the dental pieces. PMID:25047360

  12. Enzymatic Treatment of Specimens before DNA Extraction Directly Influences Molecular Detection of Infectious Agents

    PubMed Central

    Goldschmidt, Pablo; Degorge, Sandrine; Merabet, Lilia; Chaumeil, Christine

    2014-01-01

    Introduction Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. Methods Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). Results Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. Discussion Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis. PMID:24936792

  13. Fast parallel detection of feline panleukopenia virus DNA by multi-channel microchip electrophoresis with programmed step electric field strength.

    PubMed

    Nan, He; Yoo, Dong Jin; Kang, Seong Ho

    2013-01-01

    A multi-channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge-coupled device camera were used to simultaneously detect the separations in three parallel channels using laser-induced fluorescence detection. The parallel separations of a 100-bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M(r) = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single-run and one-step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi-channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease-related DNA fragments in parallel with high speed, throughput, and accuracy. PMID:23233436

  14. Attempted DNA extraction from a Rancho La Brea Columbian mammoth (Mammuthus columbi): prospects for ancient DNA from asphalt deposits

    PubMed Central

    Gold, David A; Robinson, Jacqueline; Farrell, Aisling B; Harris, John M; Thalmann, Olaf; Jacobs, David K

    2014-01-01

    Fossil-bearing asphalt deposits are an understudied and potentially significant source of ancient DNA. Previous attempts to extract DNA from skeletons preserved at the Rancho La Brea tar pits in Los Angeles, California, have proven unsuccessful, but it is unclear whether this is due to a lack of endogenous DNA, or if the problem is caused by asphalt-mediated inhibition. In an attempt to test these hypotheses, a recently recovered Columbian mammoth (Mammuthus columbi) skeleton with an unusual pattern of asphalt impregnation was studied. Ultimately, none of the bone samples tested successfully amplified M.?columbi DNA. Our work suggests that reagents typically used to remove asphalt from ancient samples also inhibit DNA extraction. Ultimately, we conclude that the probability of recovering ancient DNA from fossils in asphalt deposits is strongly (perhaps fatally) hindered by the organic compounds that permeate the bones and that at the Rancho La Brea tar pits, environmental conditions might not have been ideal for the general preservation of genetic material. PMID:24634719

  15. An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

    Microsoft Academic Search

    A. Karakousis; L. Tan; D. Ellis; H. Alexiou; P. J. Wormald

    2006-01-01

    To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods

  16. A RAPID DNA EXTRACTION METHOD IS SUCCESSFULLY APPLIED TO ITS-RFLP ANALYSIS OF MYCORRHIZAL ROOT TIPS

    EPA Science Inventory

    A rapid method for extracting DNA from intact, single root tips using a Xanthine solution was developed to handle very large numbers of analyses of ectomycorrhizas. By using an extraction without grinding we have attempted to bias the extraction towards the fungal DNA in the man...

  17. Single step plasmid DNA purification using methacrylate monolith bearing combination of ion-exchange and hydrophobic groups.

    PubMed

    Smrekar, Vida; Smrekar, Franc; Strancar, Aleš; Podgornik, Aleš

    2013-02-01

    Purification of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7 mg/ml under IEX conditions and 2.1mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5mg of pDNA/ml of monolith from cell lysate. PMID:23305854

  18. Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization

    PubMed Central

    Novak, P; Jensen, TJ; Garbe, JC; Stampfer, MR; Futscher, BW

    2009-01-01

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16 INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending upon how stasis was overcome. A second step coincides with immortalization, and results in hundreds of additional DNA methylation changes, regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. PMID:19509227

  19. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure.

    PubMed

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  20. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  1. Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

    PubMed

    Benítez, Jaime J; Topolancik, Juraj; Tian, Harvey C; Wallin, Christopher B; Latulippe, David R; Szeto, Kylan; Murphy, Patrick J; Cipriany, Benjamin R; Levy, Stephen L; Soloway, Paul D; Craighead, Harold G

    2012-11-21

    We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level. PMID:23018789

  2. Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells†

    PubMed Central

    Benítez, Jaime J.; Topolancik, Juraj; Tian, Harvey C.; Wallin, Christopher B.; Latulippe, David R.; Szeto, Kylan; Murphy, Patrick J.; Cipriany, Benjamin R.; Levy, Stephen L.; Soloway, Paul D.; Craighead, Harold G.

    2014-01-01

    We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for offchip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level. PMID:23018789

  3. Comparison of customized spin-column and salt-precipitation finger-prick blood DNA extraction.

    PubMed

    Poh, Jun-Jie; Gan, Samuel Ken-En

    2014-01-01

    gDNA (genomic DNA extraction from blood is a fundamental process in many diagnostic, identification and research applications. Numerous extraction methods have been reported and are available commercially. However, there is insufficient understanding of the impact of chemical buffers on DNA yield from either whole or nucleated blood. Moreover, these commercial kits are often costly, constraining less well-funded laboratories to traditional and more cost-effective salt-precipitation methods. Towards this, we compared a salt-precipitation and a customized cost-effective spin-column-based method, studying the impact of different chemical constituents on the yields. This customized method resulted in a shortening of the extraction process, higher gDNA yields, and more successful PCR amplification of gDNA genes compared with the salt-precipitation method. Optimizing different chemical buffers on whole- and nucleated blood materials further revealed that certain chemicals boosted extractions from whole- but not nucleated blood. These findings may be useful to laboratories that do not have ready access to commercial kits, and improve their nucleic acid extractions from blood economically. PMID:25222694

  4. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  5. High-throughput DNA extraction for PCR-based genotyping of single Penaeus monodon embryos and nauplii

    Microsoft Academic Search

    Min Rao; Stuart J. Arnold; Jeff A. Cowley

    2010-01-01

    A simple technique is described for isolating DNA from individual embryos or nauplii of the Black Tiger shrimp, Penaeus monodon. The protocol used a commercial prepGEM™ insect DNA extraction kit in combination with water bath ultrasonic disruption. DNA quality and yields were sufficient for multiplex PCR discrimination of up to six DNA microsatellites in a single test. The utility of

  6. “Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

    NASA Astrophysics Data System (ADS)

    Berthelot, Thomas; Garcia, Alexandre; Le, Xuan Tuan; El Morsli, Jenna; Jégou, Pascale; Palacin, Serge; Viel, Pascal

    2011-02-01

    Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H2O2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

  7. CENTRIFUGAL LABTUBE FOR FULLY AUTOMATED DNA EXTRACTION & LAMP AMPLIFICATION BASED ON AN INTEGRATED, LOW-COST HEATING SYSTEM

    E-print Network

    Hoehl, Melanie Margarete

    In this paper, we introduce a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA-extraction platform (LabTube). We demonstrate fully automated, ...

  8. A Fast One Step Extraction and UPLC-MS/MS Analysis for E2/D2 Series Prostaglandins and Isoprostanes

    PubMed Central

    Brose, Stephen A.; Baker, Andrew G.; Golovko, Mikhail Y.

    2013-01-01

    Prostaglandins (PG) and isoprostanes (iso-PG) may be derived through cyclooxygenase or free radical pathways and are important signaling molecules that are also robust biomarkers of oxidative stress. Their quantification is important for understanding many biological processes where PG, iso-PG, or oxidative stress are involved. One of the common methods for PG and iso-PG quantifications is LC-MS/MS that allows a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the currently used LC-MS/MS methods require a multi-step extraction and a long (within an hour) LC separation to achieve simultaneous separation and analysis of the major iso-PG. The developed and validated for brain tissue analysis one-step extraction protocol and UPLC-MS/MS method significantly increases the recovery of the PG extraction up to 95%, and allows for a much faster (within 4 min) major iso-PGE2 and -PGD2 separation with 5 times narrower chromatographic peaks as compared to previously used methods. In addition, it decreases the time and cost of analysis due to one-step extraction approach performed in disposable centrifuge tubes. All together, this significantly increases the sensitivity, and the time and cost efficiency of the PG and iso-PG analysis. PMID:23400687

  9. TECHNICAL NOTE Open Access Optimized microbial DNA extraction from

    E-print Network

    Paris-Sud XI, Université de

    pathogens reportedly causing 50% of cases of diarrhea [2]. As most of these pathogens are fastidious in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA 28.78 ± 9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04 ± 5.5); bacterial 16S r

  10. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    NASA Astrophysics Data System (ADS)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ? 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

  11. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil?†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

  12. A high-throughput plant DNA extraction method for marker analysis

    Microsoft Academic Search

    Angelo Karakousis; Peter Langridge

    2003-01-01

    The use of molecular markers to improve crops depends on the availability of rapid and efficient DNA extraction methods. Here\\u000a we describe a simple and inexpensive method to isolate plant DNA suitable for RFLP, AFLP, and simple sequence repeat (SSR)\\u000a analysis. This procedure uses stainless steel ball bearings to grind 16 samples simultaneously using a high-speed flask shaker.\\u000a The method

  13. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues

    Microsoft Academic Search

    Scott O. Rogers; Arnold J. Bendich

    1985-01-01

    We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types

  14. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    SciTech Connect

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  15. Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid?

    PubMed Central

    Yera, H.; Filisetti, D.; Bastien, P.; Ancelle, T.; Thulliez, P.; Delhaes, L.

    2009-01-01

    Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis. PMID:19846633

  16. Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

    PubMed

    Mikaeili, F; Kia, E B; Sharbatkhori, M; Sharifdini, M; Jalalizand, N; Heidari, Z; Zarei, Z; Stensvold, C R; Mirhendi, H

    2013-06-01

    Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. PMID:23499880

  17. Impact of Enhanced Staphylococcus DNA Extraction on Microbial Community Measures in Cystic Fibrosis Sputum

    PubMed Central

    Zhao, Jiangchao; Carmody, Lisa A.; Kalikin, Linda M.; Li, Jun; Petrosino, Joseph F.; Schloss, Patrick D.; Young, Vincent B.; LiPuma, John J.

    2012-01-01

    Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF. PMID:22412992

  18. Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk.

    PubMed

    Usman, T; Yu, Y; Liu, C; Fan, Z; Wang, Y

    2014-01-01

    Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280 nm and 260/230 nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows. PMID:24841664

  19. Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples

    PubMed Central

    2011-01-01

    Background Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. Methods Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses. Results The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 ?g DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. Conclusions Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses. PMID:22182470

  20. How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

    PubMed Central

    Särkinen, Tiina; Staats, Martijn; Richardson, James E.; Cowan, Robyn S.; Bakker, Freek T.

    2012-01-01

    Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL(UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200–300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies. PMID:22952770

  1. Study of partitioning and dynamics of metals in contaminated soil using modified four-step BCR sequential extraction procedure

    Microsoft Academic Search

    Tiberiu Frentiu; Michaela Ponta; Erika Levei; Emil A. Cordos

    2009-01-01

    The modified four-step BCR sequential extraction procedure (exchangeable and weak acid available species, reducible, oxidisable\\u000a and residual fractions) was used to examine the distribution of As, Cd, Cr, Cu, Pb, and Zn with soil depth in an area (Baia\\u000a Mare — Bozanta, Romania) with both high natural level of elements considered as toxic and historical pollution resulting from\\u000a nonferrous metallurgy.

  2. High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers

    Microsoft Academic Search

    Aamir Razaq; Gustav Nyström; Maria Strømme; Albert Mihranyan; Leif Nyholm

    2011-01-01

    Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg?1, were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30–50 nm conformal PPy layer

  3. Ring Trial Validation of a Method for the Extraction of DNA from Soy Lecithins

    Microsoft Academic Search

    H.-U. Waiblinger; B. Ernst; N. Graf; K. Pietsch

    2007-01-01

    :  In this work an approach for ring trial validation of a DNA extraction method for further GMO analysis is described. Practical\\u000a LOD was used as a key tool for assessing the suitability of the extraction method. In ring trial with soy lecithins, satisfactory\\u000a results with practical LODs below 0.9 % were achieved in 4 out of 5 samples. In-house tests

  4. The Effect of a Grape Seed Extract on Radiation-Induced DNA Damage in Human Lymphocytes

    NASA Astrophysics Data System (ADS)

    Dicu, Tiberius; Postescu, Ion D.; Fori?, Vasile; Brie, Ioana; Fischer-Fodor, Eva; Cernea, Valentin; Moldovan, Mircea; Cosma, Constantin

    2009-05-01

    Plant-derived antioxidants due to their phenolic compounds content are reported as potential candidates for reducing the levels of oxidative stress in living organisms. Grape seed extracts are very potent antioxidants and exhibit numerous interesting pharmacologic activities. Hydroethanolic (50/50, v/v) standardized extract was obtained from red grape seed (Vitis vinifera, variety Burgund Mare—BM). The total polyphenols content was evaluated by Folin-Ciocalteu procedure and expressed as ?Eq Gallic Acid/ml. The aim of this study was to evaluate the potential antioxidant effects of different concentrations of BM extract against 60Co ?-rays induced DNA damage in human lymphocytes. Samples of human lymphocytes were incubated with BM extract (12.5, 25.0 and 37.5 ?Eq GA/ml, respectively) administered at 30 minutes before in vitro irradiation with ?-rays (2 Gy). The DNA damage and repair in lymphocytes were evaluated using alkaline comet assay. Using the lesion score, the radiation-induced DNA damage was found to be significantly different (p<0.05) from control, both in the absence and presence of BM extract (except the lymphocytes treated with 37.5 ?Eq GA/ml BM extract). DNA repair analyzed by incubating the irradiated cells at 37° C and 5% CO2 atmosphere for 2 h, indicated a significant difference (p<0.05) in the lymphocytes group treated with 25.0 ?Eq GA/ml BM extract, immediately and two hours after irradiation. These results suggest radioprotective effects after treatment with BM extract in human lymphocytes.

  5. Determination of ABO genotypes with DNA extracted from formalin-fixed, paraffin-embedded tissues

    Microsoft Academic Search

    Mitsuko Yamada; Yoshio Yamamoto; Akio Tanegashima; Masateru Kane; Yuzuru Ikehara; Tatsushige Fukunaga; Katsuji Nishi

    1994-01-01

    Summary The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS\\/proteinase K treatment followed by phenol\\/chloroform extraction. The

  6. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

    Microsoft Academic Search

    Sue Porebski; L. Grant Bailey; Bernard R. Baum

    1997-01-01

    A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large\\u000a quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of\\u000a these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt\\u000a concentrations to remove polysaccharides, the use of polyvinyl

  7. Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification

    Microsoft Academic Search

    I. Pfeiffer; I. Völkel; H. Täubert; B. Brenig

    2004-01-01

    The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog

  8. Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts

    PubMed Central

    Knipscheer, Puck; Räschle, Markus; Schärer, Orlando D.; Walter, Johannes C.

    2014-01-01

    Summary Interstrand crosslinks (ICL) are one of the most hazardous types of DNA damage as they form a roadblock to all processes that involve strand separation. Repair of these lesions involves several different DNA repair pathways but the molecular mechanism is unclear. Here we describe a system that allows the examination of ICL repair, via a physiological mechanism, in vitro. This system, which uses Xenopus egg extracts in combination with a DNA template that contains a site-specific ICL, represents a unique tool to study the molecular mechanism of ICL repair. PMID:22941607

  9. DNA Extraction Methods for Panbacterial and Panfungal PCR Detection in Intraocular Fluids.

    PubMed

    Mazoteras, Paloma; Bispo, Paulo José Martins; Höfling-Lima, Ana Luisa; Casaroli-Marano, Ricardo P

    2014-10-01

    Abstract Purpose: Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. Material and Methods: Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. Results: The column-based extraction method was the most time-effective, achieving DNA detection limits ?10(2) and 10(3?)CFU/100?µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1?pg and 10?pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n?=?7) of patients and Staphylococcus spp. found in 22.2% (n?=?4). Conclusions: The column-based extraction method for very small inocula in small volume samples (50-100?µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them. PMID:25285466

  10. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method.

    PubMed

    Guo, Shanshan; Deng, Qianchun; Xiao, Junsong; Xie, Bijun; Sun, Zhida

    2007-04-18

    The antioxidant activities of three parts (peel, juice, and seed) and extracts of three pomegranate varieties in China were investigated by using a chemiluminescence (CL) method in vitro. The scavenging ability of pomegranate extracts (PEs) on superoxide anion, hydroxide radical, and hydrogen peroxide was determined by the pyrogallol-luminol system, the CuSO4-Phen-Vc-H2O2 system, and the luminol-H2O2 system, respectively. DNA damage preventing the effect of PE was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that the peel extract of red pomegranate had the best effect on the scavenging ability of superoxide anion because its IC50 value (4.01 +/- 0.09 microg/mL) was the lowest in all PEs. The seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the IC50 value was 1.69 +/- 0.03 microg/mL). The peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide, which had the lowest IC50 value (0.032 +/- 0.003 microg/mL) in the nine extracts. The seed extract of white pomegranate (the IC50 value was 3.67 +/- 0.03 microg/mL) was the most powerful on the DNA damage-preventing effect in all of the PEs. Also, the statistical analysis indicated that there were significant differences (at P < 0.05) among the extracts of the different varieties and parts in each system. PMID:17381116

  11. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-05-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. PMID:1349899

  12. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

  13. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

    PubMed Central

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  14. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

    PubMed

    Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  15. Steps and Bumps: Precision Extraction of Discrete States of Molecular Machines

    E-print Network

    Steel, Bradley C.

    We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical ...

  16. Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

    Microsoft Academic Search

    Caroline E. Shamu; Andrew W. Murray

    1992-01-01

    We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPE Addition of calcium induces the in- activation of MPF, sister

  17. Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes.

    PubMed

    Larsen, Andrea M; Mohammed, Haitham H; Arias, Covadonga R

    2015-03-01

    This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20°C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis. PMID:25757730

  18. Kemp Lab Ancient DNA Extraction Protocol-"New" Method Prepared by Brian M. Kemp, September 2012

    E-print Network

    Kemp, Brian M.

    Kemp Lab Ancient DNA Extraction Protocol- "New" Method Prepared by Brian M. Kemp, September 2012 Members of the Kemp Lab have come to affectionately call this protocol the "new" method, in contrast to the "old" method as described by Kemp and colleagues (2007). However, please see the updated "old" method

  19. A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

    Microsoft Academic Search

    Hee Wan Kang; Yong Gu Cho; Ung Han Yoon; Moo Young Eun

    1998-01-01

    A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be

  20. Improved DNA Extraction Method for Porcine Contaminants, Detection in Imported Meat to The Saudi Market

    Microsoft Academic Search

    Ibrahim Abdullah Alaraidh

    A porcine detection methodology based on deoxyribonucleic acid (DNA) extraction and polymerase chain reaction (PCR) amplification of a specific porcine fragment was used in this paper. With the advent of mass globalization and the fast growing and rapidly changing halal industry of the international market it is of vital need that a practical scientific system be applied and established in

  1. Near-quantitative extraction of genomic DNA from various food-borne eubacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70E6 (± 9.05%), 4.77E8 (± 31.0%), and 5.93E8 (± 4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt), Shigella sonnei (Ss), and Esch...

  2. Evaluation of a rapid DNA extraction method to detect yeast cells by PCR in orange juice

    Microsoft Academic Search

    Maria Ros-Chumillas; Marcos Egea-Cortines; Antonio Lopez-Gomez; Julia Weiss

    2007-01-01

    Yeasts are the main causes of spoilage in pasteurized orange juice. Whereas detection by plate counting techniques is too slow to allow appropriate intervention measures, PCR reaction offers a rapid alternative, but it can be inhibited by components of food samples. We developed a DNA extraction method directly from orange juice for rapid yeast detection by PCR amplification of the

  3. Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction

    Microsoft Academic Search

    Janet Cox-Singh; Suraya Mahayet; Mohd Shukri Abdullah; Balbir Singh

    1997-01-01

    Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we

  4. Detection of Genetically Modified Organisms in Food: Comparison Among Three Different DNA Extraction Methods

    Microsoft Academic Search

    B. Vodret; M. Milia; M. G. Orani; G. Serratrice; M. R. Mancuso

    2007-01-01

    Vodret, B., Milia, M., Orani, M.G., Serratrice, G. and Mancuso, M.R., 2007. Detection of genetically modified organisms in\\u000a food: Comparison among three different DNA extraction methods. Veterinary Research Communications, 31(Suppl. 1), 385–388

  5. Nucleosome assembly in mammalian cell extracts before and after DNA replication.

    PubMed Central

    Gruss, C; Gutierrez, C; Burhans, W C; DePamphilis, M L; Koller, T; Sogo, J M

    1990-01-01

    Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2167837

  6. An Improved, Single Step Standardized Method of Lipid Extraction from Human Skeletal Muscle Tissue

    Microsoft Academic Search

    Niraj Kumar Srivastava; Sunil Pradhan; Balraj Mittal; Raj Kumar; G. A. Nagana Gowda

    2006-01-01

    A new method of lipid extraction from human skeletal muscle is presented. The quantitative extraction of lipid metabolites such as phospholipids, triglycerides and cholesterol, simultaneously, in a single experiment is evaluated by H?NMR spectroscopy. Thirty?seven normal cryopreserved muscle samples were pulverized at liquid nitrogen temperature and treated with methanol and chloroform at different ratios (i.e., 1?2, 1?3, and 1?4, by

  7. Effect of Black Seed Extract (Nigella sativa) on Growth Performance, Blood Parameters, Oxidative Stress and DNA Damage of Partridges

    Microsoft Academic Search

    Mehmet Çetin; Sabri Yurtseven; Turgay ?engül; Bünyamin Sögüt

    2008-01-01

    Çetin, M., Yurtseven, S., ?engiil, T. and Sögüt, B. 2008. Effect of black seed extract (Nigella sativa) on growth performance, blood parameters, oxidative stress and DNA damage of partridges. J. Appl. Anim. Res., 34: 121–125.This study was conducted to evaluate the effect of different doses of black seed extract on fattening performance, some blood parameters, oxidative stress and DNA damage

  8. An economical and effective high-throughput DNA extraction protocol for molecular marker analysis in honey bees

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraction of DNA from tissue samples can be expensive both in time and monetary resources and can often require handling and disposal of hazardous chemicals. We have developed a high throughput protocol for extracting DNA from honey bees that is of a high enough quality and quantity to enable hundr...

  9. Improved DNA extraction method for Verticillium detection and quantification in large-scale studies using PCR-based techniques

    Microsoft Academic Search

    R. A. Heinz; H. W. Platt

    2000-01-01

    An improved plant and soil DNA extraction method for detection and quantification of Verticillium species using polymerase chain reaction (PCR) based techniques is presented. This method involves the use of extraction buffer containing proteinase K and further DNA purification with addition of ammonium acetate. In the case of soils, the protocol combines the benefits of using a commonly reported nucleic

  10. A SIMPLE METHOD FOR EXTRACTION AND PURIFICATION OF GENOMIC DNA FROM DRIED BLOOD SPOTS ON FILTER PAPER

    Microsoft Academic Search

    Sumonta Chaisomchit; Wiyada Chareonsiriwatana

    2003-01-01

    We have developed an efficient and simple method for extracting and purifying genomic DNA from dried blood stored on filter paper. The quality of the genomic DNA extracted is tested by PCR amplification of a 255-bp fragment of the PAX8 gene sequence and the PCR products are deter- mined for further genetic studies by single strand conformation polymorphism (SSCP) analysis.

  11. SHORT REPORT: RAPID DNA EXTRACTION FROM ARCHIVE BLOOD SPOTS ON FILTER PAPER FOR GENOTYPING OF PLASMODIUM FALCIPARUM

    Microsoft Academic Search

    SANDOR BERECZKY; ANDREAS MÅRTENSSON; J. PEDRO GIL; ANNA FARNERT

    2005-01-01

    The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive, and cost-effective DNA extraction methods. We describe a new Tris- EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The method was evaluated against the commonly used methanol and Chelex methods,

  12. DNA double strand break repair enzymes function at multiple steps in retroviral infection

    Microsoft Academic Search

    Yasuteru Sakurai; Kenshi Komatsu; Kazunaga Agematsu; Masao Matsuoka

    2009-01-01

    BACKGROUND: DNA double strand break (DSB) repair enzymes are thought to be necessary for retroviral infection, especially for the post-integration repair and circularization of viral cDNA. However, the detailed roles of DSB repair enzymes in retroviral infection remain to be elucidated. RESULTS: A GFP reporter assay showed that the infectivity of an HIV-based vector decreased in ATM- and DNA-PKcs-deficient cells

  13. Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

    USGS Publications Warehouse

    Steele, A.N.; Simovich, M.A.; Pepino, D.; Schroeder, K.M.; Vandergast, A.G.; Bohonak, A.J.

    2009-01-01

    The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for "DNA Barcoding", microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts. ?? Springer Science+Business Media B.V. 2008.

  14. An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region

    PubMed Central

    Fatima, Faria

    2014-01-01

    The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120?mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method. PMID:25302120

  15. Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA

    PubMed Central

    Dans, Pablo Daniel; Faustino, Ignacio; Battistini, Federica; Zakrzewska, Krystyna; Lavery, Richard; Orozco, Modesto

    2014-01-01

    We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3?-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change. PMID:25223784

  16. Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues

    PubMed Central

    Parrilla-Doblas, Jara Teresa; Ponferrada-Marín, María Isabel; Roldán-Arjona, Teresa; Ariza, Rafael R.

    2013-01-01

    Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC. PMID:23868090

  17. 762 shorT commUnicaTions AN IMPROVED EXTRACTION METHOD TO INCREASE DNA YIELD FROM MOLTED FEATHERS

    E-print Network

    - vasive source of DNA for genetic studies of Northern Goshawks (Accipiter gentilis), we isolated words: Accipiter gentilis, DNA extraction, DNA yield, molted feathers, noninvasive genetic sampling ADN en estudios de Accipiter gentilis, aislamos y cuantificamos ADN de plumas mudadas y comparamos la

  18. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    PubMed

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities. PMID:25775938

  19. New sorbent in the dispersive solid phase extraction step of quick, easy, cheap, effective, rugged, and safe for the extraction of organic contaminants in drinking water treatment sludge.

    PubMed

    Cerqueira, Maristela B R; Caldas, Sergiane S; Primel, Ednei G

    2014-04-01

    Recent studies have shown a decrease in the concentration of pesticides, pharmaceuticals and personal care products (PCPs) in water after treatment. A possible explanation for this phenomenon is that these compounds may adhere to the sludge; however, investigation of these compounds in drinking water treatment sludge has been scarce. The sludge generated by drinking water treatment plants during flocculation and decantation steps should get some special attention not only because it has been classified as non-inert waste but also because it is a very complex matrix, consisting essentially of inorganic (sand, argil and silt) and organic (humic substances) compounds. In the first step of this study, three QuEChERS methods were used, and then compared, for the extraction of pesticides (atrazine, simazine, clomazone and tebuconazole), pharmaceuticals (amitriptyline, caffeine, diclofenac and ibuprofen) and PCPs (methylparaben, propylparaben, triclocarban and bisphenol A) from drinking water treatment sludge. Afterwards, the study of different sorbents in the dispersive solid phase extraction (d-SPE) step was evaluated. Finally, a new QuEChERS method employing chitin, obtained from shrimp shell waste, was performed in the d-SPE step. After having been optimized, the method showed limits of quantification (LOQ) between 1 and 50 ?g kg(-1) and the analytical curves showed r values higher than 0.98, when liquid chromatography tandem mass spectrometry was employed. Recoveries ranged between 50 and 120% with RSD?15%. The matrix effect was evaluated and compensated with matrix-matched calibration. The method was applied to drinking water treatment sludge samples and methylparaben and tebuconazole were found in concentration

  20. High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers

    PubMed Central

    Razaq, Aamir; Nyström, Gustav; Strømme, Maria; Mihranyan, Albert; Nyholm, Leif

    2011-01-01

    Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg?1, were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30–50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m2 g?1) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)6, (dT)20, and (dT)40 DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules. PMID:22195031

  1. Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI)-Induced Cellular and DNA Toxicity

    PubMed Central

    Guha, Gunjan; Rajkumar, V.; Kumar, R. Ashok; Mathew, Lazar

    2011-01-01

    Hexavalent chromium Cr(VI) is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI)-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae), commonly known as Henna. The extracts showed significant (P < .05) potential in scavenging free radicals (DPPH• and ABTS•+) and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI)-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma) cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05) positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI)-induced oxidative cell damage. PMID:20008460

  2. Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis.

    PubMed

    Bletz, M C; Rebollar, E A; Harris, R N

    2015-02-10

    Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens. PMID:25667331

  3. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    PubMed

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered. PMID:19789856

  4. Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

    Microsoft Academic Search

    Noem?? Ruiz-López; Enrique Mart??nez-Force; Rafael Garcés

    2003-01-01

    A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results

  5. Imaging and spectroscopic comparison of multi-step methods to form DNA arrays based on the biotin-streptavidin system.

    PubMed

    Gajos, Katarzyna; Petrou, Panagiota; Budkowski, Andrzej; Awsiuk, Kamil; Bernasik, Andrzej; Misiakos, Konstantinos; Rysz, Jakub; Raptis, Ioannis; Kakabakos, Sotirios

    2015-02-21

    Three multi-step multi-molecular approaches using the biotin-streptavidin system to contact-print DNA arrays on SiO2 surfaces modified with (3-glycidoxypropyl)trimethoxysilane are examined after each deposition/reaction step by atomic force microscopy, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry. Surface modification involves the spotting of preformed conjugates of biotinylated oligonucleotides with streptavidin onto surfaces coated with biotinylated bovine serum albumin b-BSA (approach I) or the spotting of biotinylated oligonucleotides onto a streptavidin coating, the latter prepared through a reaction with immobilized b-BSA (approach II) or direct adsorption (approach III). AFM micrographs, quantified by autocorrelation and height histogram parameters (e.g. roughness), reveal uniform coverage after each modification step with distinct nanostructures after the reaction of biotinylated BSA with streptavidin or of a streptavidin conjugate with biotinylated oligonucleotides. XPS relates the immobilization of biomolecules with covalent binding to the epoxy-silanized surface. Protein coverage, estimated from photoelectron attenuation, shows that regarding streptavidin the highest and the lowest immobilization efficiency is achieved by following approaches I and III, respectively, as confirmed by TOF-SIMS microanalysis. The size of the DNA spot reflects the contact radius of the printed droplet and increases with protein coverage (and roughness) prior to the spotting, as epoxy-silanized surfaces are hardly hydrophilic. Representative TOF-SIMS images show sub-millimeter spots: uniform for approach I, doughnut-like (with a small non-zero minimum) for approach II, both with coffee-rings or peak-shaped for approach III. Spot features, originating from pinned contact lines and DNA surface binding and revealed by complementary molecular distributions (all material, DNA, streptavidin, BSA, epoxy, SiO2), indicate two modes of droplet evaporation depending on the details of each applied approach. PMID:25535629

  6. Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

    PubMed Central

    Psifidi, Androniki; Dovas, Chrysostomos I.; Bramis, Georgios; Lazou, Thomai; Russel, Claire L.; Arsenos, Georgios; Banos, Georgios

    2015-01-01

    Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals. PMID:25635817

  7. Single step aqueous two-phase extraction for downstream processing of C-phycocyanin from Spirulina platensis.

    PubMed

    Chethana, S; Nayak, Chetan A; Madhusudhan, M C; Raghavarao, K S M S

    2015-04-01

    C-phycocyanin, a natural food colorant, is gaining importance worldwide due to its several medical and pharmaceutical applications. In the present study, aqueous two-phase extraction was shown to be an attractive alternative for the downstream processing of C-phycocyanin from Spirulina platensis. By employing differential partitioning, C-phycocyanin selectively partitioned to the polymer rich (top) phase in concentrated form and contaminant proteins to the salt rich (bottom) phase. This resulted in an increase in the product purity (without losing much of the yield) in a single step without the need of multiple processing steps. Effect of process parameters such as molecular weight, tie line length, phase volume ratio, concentration of phase components on the partitioning behavior of C-phycocyanin was studied. The results were explained based on relative free volume of the phase systems. C-phycocyanin with a purity of 4.32 and yield of about 79 % was obtained at the standardized conditions. PMID:25829627

  8. Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.

    PubMed

    Janecka, Anna; Adamczyk, Agnieszka; Gasi?ska, Anna

    2015-05-01

    A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA. PMID:25640584

  9. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR

    NASA Astrophysics Data System (ADS)

    Ansari, Aseem Z.; Chael, Mark L.; O'Halloran, Thomas V.

    1992-01-01

    POSITIVE control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase1. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition2, alterations in DNA loop structures3 or optimal positioning of the activator for interaction with polymerase4. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a represser and an Hg(II)-responsive activator of bacterial mercury-resistance genes5-7.Escherichia coliRNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the represser, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site6. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33° relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.

  10. Antioxidant Activity and Protection from DNA Damage by Water Extract from Pine (Pinus densiflora) Bark

    PubMed Central

    Jiang, Yunyao; Han, Woong; Shen, Ting; Wang, Myeong-Hyeon

    2012-01-01

    Water extract from Pinus densiflora (WPD) was investigated for its antioxidant activity and its ability to provide protection from DNA damage. A series of antioxidant assays, including a 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay, a reducing power assay, a metal-chelating assay, a superoxide radical scavenging assay, and a nitrite scavenging ability, as well as a DNA damage protection assay were performed. Total phenolic content was found to be 211.32 mg Tan/g WPD. The extract scavenged 50% DPPH free radical at a concentration of 21.35 ?g/mL. At that same concentration, the reducing power ability of WPD was higher than that of ?-tocopherol. The extract chelated 68.9% ferrous ion at the concentration of 4 mg/mL. WPD showed better nitrite scavenging effect at the lower pH. Meanwhile, WPD exhibited a strong capability for DNA damage protection at 1 mg/mL concentration. Taken together, these data suggest water extract from Pinus densiflora could be used as a suitable natural antioxidant. PMID:24471072

  11. Two-dimensional continuous wavelet transform algorithm for phase extraction of two-step arbitrarily phase-shifted interferograms

    NASA Astrophysics Data System (ADS)

    Ma, Jun; Wang, Zhaoyang; Pan, Tongyan

    2014-04-01

    By virtue of the anti-noise and anti-defect abilities, in this paper, a two-dimensional continuous wavelet transform algorithm is proposed to analyze two-step arbitrarily phase-shifted interferograms with an orthonormalization process. The novel algorithm takes the advantages of the two existing ones, and it has a remarkable ability to accurately and automatically extract full-field phase distribution from two phase-shifted interferograms where they may contain arbitrary and unknown phase shift, complex fringes with phase ambiguity, large fringe-frequency variations, noise, defects and corrupted fringes. The validity of the algorithm is demonstrated by both computer simulation and real experiments.

  12. Lipid production of Chlorella vulgaris from lipid-extracted microalgal biomass residues through two-step enzymatic hydrolysis.

    PubMed

    Zheng, Hongli; Gao, Zhen; Yin, Fengwei; Ji, Xiaojun; Huang, He

    2012-08-01

    Lipid-extracted microalgal biomass residues (LMBRs) were treated using cellulase, neutrase and alcalase in a two-step process and the resulting hydrolysates were used as a source of nutrients for the cultivation of Chlorella vulgaris under non-aerated and aerated conditions for lipid production. Aeration was favorable for cell growth and lipid accumulation and a biomass of approximately 3.28 g L(-1), lipid content of 35% and lipid productivity of 116 mg L(-1) d(-1) were obtained. Thus, the tested mode of LMBRs utilization was effective for nutrient recycling in microalgal biodiesel production. PMID:22609706

  13. Reactive Molecular Dynamics study on the first steps of DNA-damage by free hydroxyl radicals

    E-print Network

    Abolfath, Ramin M; Brabec, Thomas

    2011-01-01

    We employ a large scale molecular simulation based on bond-order ReaxFF to simulate the chemical reaction and study the damage to a large fragment of DNA-molecule in the solution by ionizing radiation. We illustrate that the randomly distributed clusters of diatomic OH-radicals that are primary products of megavoltage ionizing radiation in water-based systems are the main source of hydrogen-abstraction as well as formation of carbonyl- and hydroxyl-groups in the sugar-moiety that create holes in the sugar-rings. These holes grow up slowly between DNA-bases and DNA-backbone and the damage collectively propagate to DNA single and double strand break.

  14. Extraction of single-copy nuclear DNA from forensic specimens with a variety of postmortem histories.

    PubMed

    Evison, M P; Smillie, D M; Chamberlain, A T

    1997-11-01

    Specimens of human bone, teeth and dried blood spots from 3 months to 91 years old, with a variety of postmortem histories, were used in a comparative study of recovery of single-copy nuclear DNA sequences from forensic material. Sequences of the amelogenin and HLA-DPB1 genes were chosen for their value in sexing and identification. Sequences of the mitochondrial non-coding region V were also amplified to compare the recovery of mitochondrial and single-copy nuclear DNA. A variation of the silica method for DNA extraction was refined for application to the forensic specimens in this sample. Single-copy nuclear DNA was amplified from 100% of recent postoperative bone specimens (n = 6), 80% of forensic teeth and bone specimens (n = 10), 78% of recently extracted teeth (n = 18), 78% of exhumed bone up to 91 years old (n = 37) and 69% of 15 year old bone specimens fixed in 10% formalin (n = 20). Amelogenin sexing was correct in 85% of cases (n = 74) in which the sex of the donor had been recorded. There was no correlation between the age of the specimen and the extent of DNA preservation. PMID:9397544

  15. Step-and-scan maskless lithography for ultra large scale DNA chips

    Microsoft Academic Search

    Omar D. Negrete; Franco Cerrina

    2008-01-01

    A maskless photolithography test bed was constructed to examine the requirements for stepper-based synthesis of Ultra Large Scale DNA chips (ULS-DNA chips). The test bed is based on a microscope optical layout with a 5×reduction imaging lens and micro\\/nano controlled staging at the image plane. Spatial light modulation is enabled by a Digital Micromirror Device (Texas Instruments DMD 0.7XGA) and

  16. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against ?-Bungarotoxin

    PubMed Central

    Lauridsen, Lasse H.; Shamaileh, Hadi A.; Edwards, Stacey L.; Taran, Elena; Veedu, Rakesh N.

    2012-01-01

    Background Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. Principal Findings Herein, we present a simple one-step selection of DNA aptamers against ?-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. Conclusion We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers. PMID:22860007

  17. Extraction of DNA from mucilaginous tissues of a sea slug (Elysia chlorotica).

    PubMed

    Rumpho, M E; Mujer, C V; Andrews, D L; Manhart, J R; Pierce, S K

    1994-12-01

    Efforts to study the cellular and molecular biology of the symbiotic association between opisthobranch molluscs and algal chloroplasts have been hampered by the copious amounts of mucus produced by the animals. We report for the first time a procedure for isolating total DNA free of contaminating mucilaginous compounds from the mollusc Elysia chlorotica Gould that harbors photosynthetically active chloroplasts from the siphonaceous alga, Vaucheria litorea C. Agardh. This method involves an initial extraction of fresh or freeze-dried Elysia tissue in absolute ethanol and differential processing of the resultant two-phase pellet. Final purification by CsCl-gradient centrifugation produces high molecular weight DNA suitable for molecular analysis. PMID:7873179

  18. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    PubMed

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80°C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. PMID:25579785

  19. Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues.

    PubMed

    Ruiz-López, Noemí; Martínez-Force, Enrique; Garcés, Rafael

    2003-06-15

    A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H(4)SO(2) (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method. PMID:12758264

  20. Single-step extraction and cleanup of bisphenol A in soft drinks by hemimicellar magnetic solid phase extraction prior to liquid chromatography/tandem mass spectrometry.

    PubMed

    Yazdinezhad, Samaneh Raouf; Ballesteros-Gómez, Ana; Lunar, Loreto; Rubio, Soledad

    2013-05-17

    Hemimicelles of tetradecanoate chemisorbed onto magnetic nanoparticles (MNPs) are here proposed as a sorbent for the single-step extraction and cleanup of bisphenol A (BPA) in soft drinks. The purpose of this work was to develop a simple, rapid and low-cost sample treatment suitable to assess the human exposure to BPA from this type of high consumption food. The nanoparticles were easily coated by mixing commercially available magnetite of 20-30 nm mean particle diameter with tetradecanoate at 85°C for 30 min. The extraction/cleanup procedure involved stirring the samples (3 mL) with 200mg of tetradecanoate-coated MNPs for 20 min, isolating the sorbent with a Nd-Fe-B magnet and eluting BPA with methanol. The extraction efficiency was not influenced by salt concentrations up to 1M and pH values over the range 4-9. No cleanup of the extracts was needed, and the method proved matrix-independent. The extracts were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Quantitation was performed by internal standard calibration using BPA-(13)C12. The limit of quantitation obtained for the method, 0.03 ng mL(-1), was below the usual range of concentrations reported for BPA in soft drinks (0.1-3.4 ng mL(-1)). The proposed method was successfully applied to the determination of BPA in different samples acquired from various supermarkets in southern Spain; the concentrations found ranged from 0.066 to 1.08 ng mL(-1). Recoveries from samples spiked with 0.33 ng mL(-1) of BPA ranged from 91% to 105% with relative standard deviations from 3% to 8%. PMID:23639396

  1. Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

    PubMed Central

    Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

    2014-01-01

    Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE. PMID:24688314

  2. DNA Fingerprinting in a Forensic Teaching Experiment

    ERIC Educational Resources Information Center

    Wagoner, Stacy A.; Carlson, Kimberly A.

    2008-01-01

    This article presents an experiment designed to provide students, in a classroom laboratory setting, a hands-on demonstration of the steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analysis of the data. This experiment demonstrates how DNA fingerprinting is performed and how long it takes. It…

  3. DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees

    PubMed Central

    Bithell, Sean L.; Tran-Nguyen, Lucy T. T.; Hearnden, Mark N.; Hartley, Diana M.

    2015-01-01

    Understanding the root distribution of trees by soil coring is time-consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m?2) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23–28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R2 = 0.9307, P < 0.001) with the dry matter (g m?2) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

  4. DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees.

    PubMed

    Bithell, Sean L; Tran-Nguyen, Lucy T T; Hearnden, Mark N; Hartley, Diana M

    2014-01-01

    Understanding the root distribution of trees by soil coring is time -: consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m(-2)) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23-28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R(2) = 0.9307, P < 0.001) with the dry matter (g m(-2)) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

  5. In Vitro Effect of Aqueous Extract and Fraction IV Portion of Ximenia americana Stem Bark on Trypanosoma congolense DNA

    PubMed Central

    Maikai, Victor Ambrose; Maikai, Beatty Viv; Kobo, Patricia Ishyaku

    2014-01-01

    Trypanosomosis is a debilitating disease affecting mainly livestock and humans in tropical Africa. Chemically synthesized drugs and medicinal plants have been used in the treatment and control of this disease. In this study, the in vitro effect of aqueous extracts and fraction IV extract of Ximenia americana stem bark on Trypanosoma congolense DNA was investigated. The extracts were incubated with the parasites in vitro at 300?mg/mL aqueous extract and 25?mg/mL fraction IV portion for 30, 60, and 120 mins. The DNA of the trypanosomes was isolated and digested using ECOR1 enzyme and subsequently PCR was carried out. Results showed that aqueous extract and fraction IV portion immobilized 55% and 90% of the trypanosomes after 30-minute incubation. Subsequent isolation of the parasite DNA and agarose gel electrophoresis did not reveal that cell death was as a result of DNA fragmentation. This suggests that cell death was by another mechanism of action. PMID:24587898

  6. A simple and rapid DNA extraction method for the detection of Wuchereria bancrofti infection in the vector mosquito, Culex quinquefasciatus by Ssp I PCR assay

    Microsoft Academic Search

    V. Vasuki; S. L. Hoti; C. Sadanandane; P. Jambulingam

    2003-01-01

    A simple, rapid and inexpensive method for the extraction of DNA from filarial vector, Culexquinquefasciatus, useful in Ssp I PCR assay for xenomonitoring of infection with Wuchereriabancrofti is presented. The DNA extracted by this method was found suitable for PCR detection of W. bancrofti infection in pools of 10–30 mosquitoes. The PCR assay employing the simplified DNA extraction method was

  7. MEMORIAS DO INSTITUTO OSWALDO CRUZ 2008 vol 103 n 5 pp 501-5031 Running headline: Schistosoma mansoni DNA extraction method

    E-print Network

    Paris-Sud XI, Université de

    16 17 18 19 Running headline: Schistosoma mansoni DNA extraction method Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction

  8. DNA extraction Laboratory 1.03g, Donnan Laboratories, Crown Street, School of Biological Sciences, University of Liverpool. L69 7ZD.

    E-print Network

    Steve Kemp

    DNA extraction Laboratory 1.03g, Donnan Laboratories, Crown Street, School of Biological Sciences, University of Liverpool. L69 7ZD. Extraction of DNA from tissue: High salt method. Version 1.0, September://sciencepark.mdanderson.org/mbcore/protocols.html Any comments or suggestions should be sent to Phill Watts at p.c.watts@liv.ac.uk. #12;DNA extraction

  9. Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

    PubMed Central

    Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

    2013-01-01

    DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, ?-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the ?-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAFV600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

  10. Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR

    PubMed Central

    2014-01-01

    Introduction: Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS). Materials and Methods: Sixteen venous blood samples collected in K3-EDTA tubes (400?l of whole blood) were used for the spotting (4 circles each 100?l) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT™ dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of ß-thalassemia patients were also performed to ensure the quality of extraction. Results: The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ ?l was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/?l for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/?l when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA. Conclusion: It was concluded that the superparamagnetic-bead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes. PMID:24959449

  11. A magnetic nanoparticles-based method for DNA extraction from the saliva of stroke patients

    PubMed Central

    Yi, Li; Huang, Ying; Wu, Ting; Wu, Jun

    2013-01-01

    C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is a risk factor for stroke, suggesting that widespread detection could help to prevent stroke. DNA from 70 stroke patients and 70 healthy controls was extracted from saliva using a magnetic nanoparticles-based method and from blood using conventional methods. Real-time PCR results revealed that the C677T polymorphism was genotyped by PCR using DNA extracted from both saliva and blood samples. The genotype results were confirmed by gene sequencing, and results for saliva and blood samples were consistent. The mutation TT genotype frequency was significantly higher in the stroke group than in controls. Homocysteine levels were significantly higher than controls in both TT genotype groups. Therefore, this noninvasive magnetic nanoparticles-based method using saliva samples could be used to screen for the MTHFR C677T polymorphism in target populations. PMID:25206624

  12. Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction

    Microsoft Academic Search

    Leslie A. Dauphin; Roblena E. Walker; Jeannine M. Petersen; Michael D. Bowen

    2011-01-01

    This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure

  13. Evaluation of DNA Extraction Methods for Molecular Analyses of Microbial Communities in Modern Calcareous Microbialites

    Microsoft Academic Search

    Brian D. Wade; Ferran Garcia-Pichel

    2003-01-01

    We evaluated and optimized three rapid methods for extraction of high-quality DNA from carbonate-encrusted microbial communities using modern calcifying oncolites built by cyanobacteria and diatoms in a high-calcium freshwater river. Pulverization, acid (HCl) dissolution, and chelator-mediated (EDTA) dissolution of the carbonate matrix were used and optimized to liberate microbial cells from their mineral encasing. This was followed by cell lysis

  14. Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay

    Microsoft Academic Search

    Yoo Kyoung Park; Hyang Burm Lee; Eun-Jae Jeon; Hack Sung Jung; Myung-Hee Kanga

    2004-01-01

    The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in

  15. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

    PubMed

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H C; De Keersmaecker, Sigrid C J

    2014-12-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  16. Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

    PubMed Central

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

    2014-01-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at ?20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  17. Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.

    PubMed

    Vo, A-T E; Jedlicka, J A

    2014-11-01

    Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing. PMID:24774752

  18. Inhibitory activities of microalgal extracts against Epstein-Barr virus DNA release from lymphoblastoid cells.

    PubMed

    Kok, Yih-Yih; Chu, Wan-Loy; Phang, Siew-Moi; Mohamed, Shar Mariam; Naidu, Rakesh; Lai, Pey-Jiun; Ling, Shui-Nyuk; Mak, Joon-Wah; Lim, Patricia Kim-Chooi; Balraj, Pauline; Khoo, Alan Soo-Beng

    2011-05-01

    This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitt's lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC(50)<0.01 µg/ml) with a high therapeutic index (>28000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC(50)=2.9 µg/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1'a was most active in reducing the cell-free EBV DNA (EC(50)=1.38 µg/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV. PMID:21528487

  19. Inhibitory activities of microalgal extracts against Epstein-Barr virus DNA release from lymphoblastoid cells*

    PubMed Central

    Kok, Yih-Yih; Chu, Wan-Loy; Phang, Siew-Moi; Mohamed, Shar Mariam; Naidu, Rakesh; Lai, Pey-Jiun; Ling, Shui-Nyuk; Mak, Joon-Wah; Lim, Patricia Kim-Chooi; Balraj, Pauline; Khoo, Alan Soo-Beng

    2011-01-01

    This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitt’s lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC50<0.01 µg/ml) with a high therapeutic index (>28 000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC50=2.9 µg/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1’a was most active in reducing the cell-free EBV DNA (EC50=1.38 µg/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV. PMID:21528487

  20. Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities

    Microsoft Academic Search

    Ann H. Y. Kwan; Robert Czolij; Joel P. Mackay; Merlin Crossley

    2003-01-01

    The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We des- cribe a strategy for determining whether novel proteins possess typical sequence-specific DNA- binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 45 possible 5 bp sites, one might expect the

  1. Initial steps towards a production platform for DNA sequence analysis on the grid

    Microsoft Academic Search

    Angela C. M. Luyf; Barbera D. C. van Schaik; Michel de Vries; Frank Baas; Antoine H. C. van Kampen; Sílvia D. Olabarriaga

    2010-01-01

    BACKGROUND: Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids

  2. [Determination of levamisole residue in animal livers by two liquid-liquid extraction steps-gas chromatography-mass spectrometry].

    PubMed

    Xu, Jing; Xiao, Shanshan; Dong, Weifeng; Sui, Kai; Cao, Jijuan; Diao, Wenting; Zhang, Jing

    2012-09-01

    A novel method wasdeveloped for the determination oflevamisole in animal livers by liquid-liquid extraction and gas chromatography-mass spectrometry. Levamisole hydrochloride was transferred to levamisole in alkalinesolution, andthen extracted intoethyl acetate. Two liquid-liquid extraction steps were carried out with HCI aqueous solution and potassium hydroxide-dichloromethane system in turn, and the liposoluble substances and the water-soluble substances were removed, respectively. The qualitative and quantitative analyses of levamisole were achieved by gas chromatography-mass spectrometry (GC-MS) system. The characteristic fragments m/z 148, 176 and 204 were selected and m/z 204 was used as the quantitative ion. A good linear relationship was obtained between the peak area and the mass concentration of levamisole from 0.25 mg/L to 3.0 mg/L with the correlation coefficient of 0.999. The limit of quantitation (LOQ) for levamisole was 5 microg/kg, which was much lower than the international maximum residue limit. The average recoveries of levamisole spiked in chicken liver, duck liver, rabbit liver and pig liver samples at three spiked levels ranged from 76% to 106% with the relative standard deviations less than 9%. The method is simple and stable without tedious sample preparation, and suitable for the determination of levamisole in animal livers. PMID:23285974

  3. Computer-based knowledge extraction tool: a step in the development of a cognitive skills tutor

    SciTech Connect

    Stoddard, M.L.; Kern, R.P.; Emerson, J.D.

    1986-01-01

    Los Alamos National Laboratory, under the sponsorship of the Army Research Institute, is developing an experimental computer-tutor to be used as part of the Armor Officer's Basic Course at Fort Knox, Kentucky. The tutor's objective is to train students to apply the types of cognitive processing strategies needed to more effectively organize their knowledge for application in planning and conducting tactical operations. The tutor is being developed through an iterative process with the first phase being a knowledge extraction computer-based tool. Student knowledge organization in this domain will be obtained through collection of online and offline performance data. The tool is designed to obtain the knowledge organization through a motivating, realistic tactical operations exercise. 18 refs.

  4. An Easy, Rapid, and Cost-Effective Method for DNA Extraction from Various Lichen Taxa and Specimens Suitable for Analysis of Fungal and Algal Strains

    PubMed Central

    Park, Sook-Young; Jang, Seol-Hwa; Oh, Soon-Ok; Kim, Jung A

    2014-01-01

    Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. PMID:25606001

  5. An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

    PubMed

    Park, Sook-Young; Jang, Seol-Hwa; Oh, Soon-Ok; Kim, Jung A; Hur, Jae-Seoun

    2014-12-01

    Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. PMID:25606001

  6. EVALUATION OF DIFFERENT METHODS FOR THE EXTRACTION OF DNA FROM FUNGAL CONIDIA BY QUANTITATIVE COMPETITIVE PCR ANALYSIS

    EPA Science Inventory

    Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartaru...

  7. Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

    SciTech Connect

    Xun Li; Traganos, F.; Melamed, M.R.; Darzynkiewicz, Z. [New York Medical College, Valhalla, NY (United States)

    1995-06-01

    The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin`s lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. 30 refs., 7 figs.

  8. Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR?

    PubMed Central

    Muñoz-Cadavid, C.; Rudd, S.; Zaki, S. R.; Patel, M.; Moser, S. A.; Brandt, M. E.; Gómez, B. L.

    2010-01-01

    DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory. PMID:20392915

  9. KRAS mutation screening by chip-based DNA hybridization - a further step towards personalized oncology.

    PubMed

    Steinbach, Christine; Steinbrücker, Carolin; Pollok, Sibyll; Walther, Katharina; Clement, Joachim H; Chen, Yuan; Petersen, Iver; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-04-21

    The use of predictive biomarkers can help to improve therapeutic options for the individual cancer patient. For the treatment of colon cancer patients with anti-EGFR-based drugs, the KRAS mutation status has to be determined to pre-select responders that will benefit from this medication. Amongst others, array-based tests have been established for profiling of the KRAS mutation status. Within this article we describe an on-chip hybridization technique to screen therapeutic relevant KRAS codon 12 mutations. The DNA chip-based platform enables the reliable discrimination of selected mutations by allele-specific hybridization. Here, silver deposits represent robust endpoint signals that allow for a simple naked eye rating. With the here presented assay concept a precise identification of heterozygous and homozygous KRAS mutations, even against a background of up to 95% wild-type DNA, was realizable. The applicability of the test was successfully proven for various cancer cell lines as well as clinical tumour samples. Thus, the chip-based DNA hybridization technique seems to be a promising tool for KRAS mutation analysis to further improve personalized cancer treatment. PMID:25706807

  10. The Effects from DNA Extraction Methods on the Evaluation of Microbial Diversity Associated with Human Colonic Tissue

    Microsoft Academic Search

    Páraic Ó Cuív; Daniel Aguirre de Cárcer; Michelle Jones; Eline S. Klaassens; Daniel L. Worthley; Vicki L. J. Whitehall; Seungha Kang; Christopher S. McSweeney; Barbara A. Leggett; Mark Morrison

    2011-01-01

    Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout\\u000a the world, and might be re-examined to better understand host–microbe interactions in health and disease. However, the published\\u000a protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their\\u000a recovery of the microbial fraction associated with

  11. Comparative analysis of soil DNA extraction methods to study various ammonium-oxidizing bacteria and archaea by PCR-DGGE

    Microsoft Academic Search

    A. S. Cherobaeva; A. L. Stepanov; I. K. Kravchenko

    2011-01-01

    A comparative analysis of five methods of extraction and purification of soil DNA, including a modification of the authors,\\u000a was performed for the further molecular investigation of various ammonium-oxidizing bacteria and archaea in soils. Experiments\\u000a using soil samples from natural ecosystems and agroecosystems of the European area of Russia established that the amount of\\u000a DNA extracted by different methods depended

  12. Step-arrest mutants of FLP recombinase: Implications for the catalytic mechanism of DNA recombination

    SciTech Connect

    Parsons, R.L.; Prasad, P.V.; Harshey, R.M.; Jayaram, M.

    1988-08-01

    The site-specific recombinase (FLP) encoded by the yeast plasmid 2..mu..m circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues. His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. The authors' report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. They provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.

  13. Determination of chloropropanols in foods by one-step extraction and derivatization using pressurized liquid extraction and gas chromatography-mass spectrometry.

    PubMed

    Racamonde, I; González, P; Lorenzo, R A; Carro, A M

    2011-09-28

    3-Chloropropane-1,2-diol (3-MCPD) and 1,3-dichloro-2-propanol (1,3-DCP) were determined for the first time in bakery foods using pressurized liquid extraction (PLE) combined with in situ derivatization and GC-MS analysis. This one-step protocol uses N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as silylation reagent. Initially, screening experimental design was applied to evaluate the effects of the variables potentially affecting the extraction process, namely extraction time (min) and temperature (°C), number of cycles, dispersant reagent (diatomaceous earth in powder form and as particulate matter with high pore volume Extrelut NT) and percent of flush ethyl acetate volume (%). To reduce the time of analysis and improve the sensitivity, derivatization of the compounds was performed in the cell extraction. Conditions, such as the volume of BSTFA, temperature and time for the in situ derivatization of analytes using PLE, were optimized by a screening design followed to a Doehlert response surface design. The effect of the in-cell dispersants/adsorbents with diatomaceous earth, Florisil and sodium sulfate anhydrous was investigated using a Box-Behnken design. Using the final best conditions, 1 g of sample dispersed with 0.1 g of sodium sulfate anhydrous and 2.5 g diatomaceous earth was extracted with ethyl acetate. 1 g of Florisil, as clean-up adsorbent, and 70 ?L of BSTFA were used for 3 min at 70°C. Under the optimum conditions, the calibration curves showed good linearity (R(2)>0.9994) and precision (relative standard deviation, RSD?2.4%) within the tested ranges. The limits of quantification for 1,3-DCP and 3-MCDP, 1.6 and 1.7 ?g kg(-1), respectively, are far below the established limits in the European and American legislations. The accuracy, precision, linearity, and limits of quantification provided make this analytical method suitable for routine control. The method was applied to the analysis of several toasted bread, snacks, cookies and cereal samples, none of which contained chloropropanols at concentrations above the legislation levels. PMID:21875707

  14. Comparison of four methods for extracting DNA from dried blood on filter paper for PCR targeting the mitochondrial Plasmodium genome

    PubMed Central

    Strøm, Gro E. A.; Tellevik, Marit G.; Hanevik, Kurt; Langeland, Nina; Blomberg, Bjørn

    2014-01-01

    Background Few studies comparing multiple methods for DNA extraction from dried blood spots (DBS) on filter paper for PCR targeting the Plasmodium genome have been done. Methods Frequently-used methods for DNA extraction from DBS using Chelex-100, InstaGene Matrix, QIAamp DNA Mini Kit and TE buffer were compared on a dilution series of a standardized Plasmodium falciparum positive sample. The two DNA extraction methods resulting in the lowest limits of detection were compared by testing both on 31 P. falciparum positive samples collected under field conditions and stored for 4 years. Results The Chelex-100, InstaGene Matrix and QIAamp DNA Mini Kit methods performed similarly, resulting in the detection of 0.5 to 2 parasites per microliter (p/µl). The same 13 clinical samples (13/31; 42%) were positive using both DNA extraction methods with the lowest limits of detection. Conclusions Simple and low-cost methods can be sensitive and useful in extracting DNA from DBS. Poor results on stored clinical DBS indicate that further studies on the impact of storage duration and conditions, and choice of filter paper should be performed. PMID:24907711

  15. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    ERIC Educational Resources Information Center

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  16. Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

    PubMed Central

    Miyata, Marcelo; Santos, Adolfo Carlos Barreto; Mendes, Natália Helena; Cunha, Eunice Atsuko; de Melo, Fernando Augusto Fiúza; Leite, Clarice Queico Fujimura

    2011-01-01

    We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping. PMID:24031692

  17. A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches.

    PubMed

    Gunnigle, Eoin; Ramond, Jean-Baptiste; Frossard, Aline; Seeley, Mary; Cowan, Don

    2014-08-01

    A co-extraction protocol that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities is presented. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA- and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction. PMID:24929037

  18. Protocol for DNA Isolation 1. Put 250 L extraction buffer in 1.5 mL Eppendorf tube.

    E-print Network

    Russell, Amy L.

    Protocol for DNA Isolation 1. Put 250 µL extraction buffer in 1.5 mL Eppendorf tube. 2. Add wing.Carefully pour off ethanol, keeping track of the DNA pellet at the bottom of the tube. 20.Dry the pellet under vacuum or on benchtop, until thoroughly dry. 21.Resuspend the DNA in 15-20 µL of 1/10 TE. 22

  19. DNA stretch during electrophoresis due to a step change in mobility.

    PubMed

    Underhill, Patrick T; Doyle, Patrick S

    2007-07-01

    We investigate DNA stretching during electrophoresis when the mobility abruptly changes. This is a simplified geometry that produces a nonhomogeneous strain rate over the scale of a single molecule. An effective Weissenberg number (Wi) and Deborah number were identified, and the degree of stretching was examined as a function of these two parameters. The system does not undergo a coil-stretch transition. The finite extensibility of the chains only affects the response if the chain is stretched to a significant fraction of the contour length. The wormlike chain shows a characteristic approach to full extension of Wi(-1/2). PMID:17677482

  20. Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis.

    PubMed

    Linton, Elisabeth; Rahman, Asif; Viamajala, Sridhar; Sims, Ronald C; Miller, Charles D

    2012-01-01

    In this study, a proton nuclear magnetic resonance (1H NMR) method was developed to quantitatively analyze polyhydroxyalkanoate (PHA) content in Cupriavidus necator H16, Azotobacter vinelandii AvOP, and mixed microbial cultures from the effluent of an agricultural waste treatment anaerobic digester. In contrast to previous methods, a single-step PHA extractive method using deuterated chloroform was established, thereby facilitating direct 1H NMR analysis. The accuracy of the method was verified through comparison with well-established gas chromatography (GC) methanolysis techniques. Nile blue fluorescence staining was also carried out to serve as an independent and qualitative indicator of intracellular PHA content. The results indicate that the 1H NMR method is appropriate for rapid and non-destructive quantification of overall PHA content and determination of PHA copolymer composition in a variety of cultures. Notably, this technique was effective in measuring PHA content in full-strength waste samples where high concentrations of background impurities and organic compounds are present. The straightforward procedures minimize error-introducing steps, require less time and materials, and result in an accurate method suitable for routine analyses. PMID:22797227

  1. Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening

    PubMed Central

    Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

    2013-01-01

    Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ?1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ?98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS. PMID:24498621

  2. Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening.

    PubMed

    Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

    2013-11-01

    Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ?1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ?98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS. PMID:24498621

  3. A one-step extraction procedure for the screening of cocaine, amphetamines and cannabinoids in postmortem blood samples.

    PubMed

    Pelição, Fabrício Souza; Peres, Mariana Dadalto; Pissinate, Jauber Fornaciari; De Martinis, Bruno Spinosa

    2014-01-01

    A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification in postmortem whole blood samples of cocaine (COC), amphetamines (AMPs) and cannabis; the main drugs involved in cases of impaired driving in Brazil. The analytes were extracted by solid-phase extraction by means of Bond-Elute Certify cartridges, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide at 80°C for 30 min and analyzed by GC-MS. Linearity ranged from 10 to 500 ng/mL, except for ecgonine methyl ester, for which linearity ranged from 10 to 100 ng/mL. Inter- and intra-day imprecision ranged from 2.8 to 18.4% and from 1.5 to 14.9%, respectively. Accuracy values lay between 86.9 and 104.4%. The limit of quantitation for all drugs was 10 ng/mL and recoveries were >74% for all analytes, except for cannabinoids, which showed poor recovery (?30%). The developed method was applied to real samples collected from deceased victims due to traffic accidents. These samples were selected according to the results obtained in immunoassay screening on collected urine samples. Five samples were positive for the presence of COC and metabolites, four samples were positive for cannabinoids, six samples were positive for AMPs and two samples were drug negative. Some samples were positive for more than one class of drug. Results obtained from whole blood samples showed good agreement with urine screening. The developed method proved capable of quantifying all three classes of drugs of abuse proposed in this study, through a one-step extraction procedure. PMID:24782143

  4. [In vitro repair of gamma-irradiated transforming Bacillus subtilis DNA by extracts of blue-green algae].

    PubMed

    Shevchenko, T N; Gushcha, N I; Dmitriev, A P; Grodzinski?, D M

    1982-04-01

    A cell-free extract from blue-green alga Anacystis nidulans contains enzymes which repair in vitro the transforming activity of gamma-irradiated Bacillus subtilis DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture, the duration of incubation and on the dose of irradiation. The repair of gamma-induced lesions is most efficient in the presence of magnesium ions, NAD and ATP. The present data indicate that the repair of transforming DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of algae. PMID:6806146

  5. Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction

    PubMed Central

    Sankaranarayanan, Krithivasan; Timofeeff, Michael N.; Spathis, Rita; Lowenstein, Tim K.; Lum, J. Koji

    2011-01-01

    Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system. PMID:21694765

  6. Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.

    PubMed

    Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

    2013-09-01

    The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H?O?-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65±0.97 mg GAE/g), total flavonoid content (8.04±0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48±0.21 mg Na?EDTA/g). PMID:23871827

  7. Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

    PubMed

    Sankaranarayanan, Krithivasan; Timofeeff, Michael N; Spathis, Rita; Lowenstein, Tim K; Lum, J Koji

    2011-01-01

    Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system. PMID:21694765

  8. Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo

    PubMed Central

    Ke, Yuebin; Xu, Xinyun; Wu, Shuang; Huang, Juan; Misra, Hara; Li, Yunbo

    2013-01-01

    Objective. To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. Results. FR was shown to effectively protect against DNA damage induced by H2O2??in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo. PMID:24089629

  9. Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

    PubMed Central

    Beyert, N; Reichenberger, S; Peters, M; Hartung, M; Göttlich, B; Goedecke, W; Vielmetter, W; Pfeiffer, P

    1994-01-01

    Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt. Images PMID:8202366

  10. Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album's ethanolic leaf extract.

    PubMed

    Elif Korcan, S; Aksoy, Onur; Erdo?mu?, S Feyza; Ci?erci, ? Hakk?; Konuk, Muhsin

    2013-01-01

    We have investigated the antibacterial effects of Chenopodium album's ethanolic leaf extract (CAE) on all the Gram (+) and Gram (-) microorganisms and evaluated the protective effects of CAE on both yeast and human mononuclear leukocytes' genomic DNA upon oxidative shock. Antibacterial activity was recorded on Bacillus subtilis with 13 mm of inhibition zone. Total oxidative status (TOS) and the total antioxidative status (TAS) levels were determined to evaluate the antioxidant activity of CAE. Results indicated that there was a good correlation between dose of CAE and TAS levels. We also observed that CAE protect the DNA of both yeast and mononuclear leukocytes against the damaging effect of hydrogen peroxide. The comet assay, applied on both Saccharomyces cerevisiae BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0) and human leukocytes, results suggested that there was statistically significant correlation between CAE dilutions and antigenotoxic activity. PMID:22897836

  11. Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

    Microsoft Academic Search

    Linda W. Chui; Robin King; Patricia Lu; Ken Manninen; Jeong Sim

    2004-01-01

    Polymerase chain reaction (PCR) has been widely used due to its high specificity, sensitivity, and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. In this study, DNA extraction methods for Mycobacterium avium subsp. paratuberculosis were evaluated including rapid lysis, organic extraction, silica-based and magnetic particle-based (MagaZorbTM) technologies

  12. Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts.

    PubMed

    Blanco, Miguel G; Boán, Francisco; Barros, Paula; Castaño, José G; Gómez-Márquez, Jaime

    2005-09-01

    We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair. PMID:16051267

  13. Influence of amount of starting material for DNA extraction on detection of low-level presence of genetically engineered traits.

    PubMed

    Demeke, Tigst; Phan, Anh; Ratnayaka, Indira; Holigroski, Michelle; Jenkins, G Ronald

    2014-05-14

    Two laboratories independently examined how the amount of starting material influences DNA extraction efficiency and, ultimately, the detection of low-level presence of genetically engineered (GE) traits in commercialized grains. GE traits from one maize, two canola, and two soybean samples were used as prototypical models in the study design as well as two commonly used DNA extraction methods, a small scale (0.1 and 0.2 g samples) and a large scale (1.0 and 2.0 g samples). The DNA samples were fortified (spiked) at 0.1 and 0.01% (w/w) levels. The amount of DNA recovery varied between the two laboratories, although a sufficient amount of DNA was obtained to perform replicate PCR analysis by both laboratories. Reliable detection of all five events was achieved by both laboratories at 0.1% level using either small-scale or large-scale DNA extractions. Reliable detection of the GE events was achieved at 0.01% level for soybean and canola but not for maize. Variability was observed among the two laboratories in terms of the Ct values generated. There was no difference between small-scale and large-scale DNA extraction methods for qualitative PCR detections of all five GE events. PMID:24745691

  14. Recovery of primary sporocysts in vivo in the Schistosoma mansoni / Biomphalaria1 glabrata model using a simple fixation method suitable for extraction of genomic DNA2

    E-print Network

    Paris-Sud XI, Université de

    using a simple fixation method suitable for extraction of genomic DNA2 and RNA.3 4 5 Jean we describe a method to circumvent this problem and to analyze DNA and28 RNA of Schistosoma mansoni for improved DNA and RNA extraction30 from the intra-molluscan stage of the parasite recovered after fixation

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  17. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn, E-mail: dc17@york.ac.uk

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  18. Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

    PubMed Central

    Podnecky, Nicole L.; Elrod, Mindy G.; Newton, Bruce R.; Dauphin, Leslie A.; Shi, Jianrong; Chawalchitiporn, Sutthinan; Baggett, Henry C.; Hoffmaster, Alex R.; Gee, Jay E.

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen. PMID:23460920

  19. Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

    PubMed Central

    Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2008-01-01

    Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ?100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

  20. Five commercial DNA extraction systems tested and compared on a stool sample collection.

    PubMed

    Persson, Søren; de Boer, Richard F; Kooistra-Smid, Anna M D; Olsen, Katharina E P

    2011-03-01

    In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis. PMID:21353945

  1. In vitro free radical scavenging and DNA damage protective property of Coriandrum sativum L. leaves extract.

    PubMed

    Harsha, S N; Anilakumar, K R

    2014-08-01

    Coriandrum sativum L. (coriander), an everyday spice in the Indian kitchen is known to add flavor to the cuisine. It is an annual herb belonging to the Apiaceae (Umbellifera) family. The hydro-alcohol extract of Coriandrum sativum L. at the dose of 1 mg/ml was subjected to a series of in vitro assays viz. 2, 2'- diphenyl-1-picrylhydrazyl, lipid peroxidation by thiobarbituric acid, reducing power and nitric oxide (NO) radical scavenging in order to study its antioxidant efficacy in detail. The amount of flavonoids in 70% ethanol extract was found to be 44.5 ?g and that of the total phenols was 133.74 ?g gallic acid equivalents per mg extract. The extracts of the leaves showed metal chelating power, with IC50 values, 368.12 ?g/ml where as that of standard EDTA was 26.7 ?g/ml. The IC50 values for 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid radical scavenging was 222 ?g/ml where as that of standard ascorbic acid was 22.6 ?g/ml. The NO scavenging activity of the extract of the leaves showed IC50 value of 815.6 ?g/ml; at the same time the standard BHA had 49.1 ?g/ml. All the plant extracts provided DNA damage protection; however, the protection provided at the dose of 8 ?g/ml was comparable to that of standard gallic acid. The Coriandrum sativum leaf extract was able to prevent in vitro lipid peroxidation with IC50 values; 589.6 ?g/ml where as that of standard BHA was 16.3 ?g/ml. Our results also showed significant ferric reducing power indicating the hydrogen donating ability of the extract. This study indicated the potential of the leaf extract as a source of natural antioxidants or nutraceuticals that could be of use in food industry with potential application to reduce oxidative stress in living system. PMID:25114344

  2. Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics

    PubMed Central

    Bongini, Lorenzo; Melli, Luca; Lombardi, Vincenzo; Bianco, Pasquale

    2014-01-01

    Under a tension of ?65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the ?-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

  3. Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.

    PubMed

    Bongini, Lorenzo; Melli, Luca; Lombardi, Vincenzo; Bianco, Pasquale

    2014-03-01

    Under a tension of ?65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the ?-phage DNA with different melting degrees and temperatures (10-25°C). The shortening transient following a 20-35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2-35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

  4. Determination of gold, indium, tellurium and thallium in the same sample digest of geological materials by atomic-absorption spectroscopy and two-step solvent extraction

    USGS Publications Warehouse

    Hubert, A.E.; Chao, T.T.

    1985-01-01

    A rock, soil, or stream-sediment sample is decomposed with hydrofluoric acid, aqua regia, and hydrobromic acid-bromine solution. Gold, thallium, indium and tellurium are separated and concentrated from the sample digest by a two-step MIBK extraction at two concentrations of hydrobromic add. Gold and thallium are first extracted from 0.1M hydrobromic acid medium, then indium and tellurium are extracted from 3M hydrobromic acid in the presence of ascorbic acid to eliminate iron interference. The elements are then determined by flame atomic-absorption spectrophotometry. The two-step solvent extraction can also be used in conjunction with electrothermal atomic-absorption methods to lower the detection limits for all four metals in geological materials. ?? 1985.

  5. Comparative evaluation of Amplicor HIV-1 DNA test, version 1.5, by manual and automated DNA extraction methods using venous blood and dried blood spots for HIV-1 DNA PCR testing.

    PubMed

    Nsojo, Anthony; Aboud, Said; Lyamuya, Eligius

    2010-10-01

    Human immunodeficiency virus (HIV) DNA polymerase chain reaction (PCR) test using venous blood sample has been used for many years in low resource settings for early infant diagnosis of HIV infection in children less than 18 months. The aim of this study was to evaluate and compare the performance characteristics of Amplicor HIV-1 DNA assay version 1.5 following processing of venous blood and dried blood spot (DBS) samples by Roche manual DNA extraction and automated Roche MagNA Pure LC instrument (MP) for HIV-1 DNA PCR testing in Dar es Salaam, Tanzania, in order to scale up early infant diagnosis of HIV infection in routine practice. Venous blood samples from children under 18 months born to HIV-infected mothers between January and April 2008 were collected. Venous blood was used to prepare cell pellet and DBS samples. DNA extractions by manual procedure and MP were performed each on cell pellet, venous blood and DBS samples and tested by Amplicor HIV-1 DNA assay. Of 325 samples included, 60 (18.5%) were confirmed HIV-infected by manual extraction performed on cell pellets. Sensitivity of the assay following MP processing of venous blood was 95% (95% CI; 86.1-99.0%) and 98.3% (95% CI; 91.1 to 99.9%) for the manual extraction and processing by MP performed on DBS samples. Specificity of the assay with all DNA extraction methods was 99.6% (95% CI; 97.9 to 100%). Performance of the assay with Roche manual extraction and processing by MP on DBS samples compared well with Roche manual extraction performed on cell pellet samples. The choice of DNA extraction method needs to be individualized based on the level of laboratory facility, volume of testing and cost benefit analysis before it is adopted for use. PMID:24409629

  6. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

    PubMed Central

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stéphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  7. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition.

    PubMed

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stéphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  8. Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

    NASA Astrophysics Data System (ADS)

    Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

    2010-12-01

    Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

  9. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  10. A rapid typing system at three STR loci from bloodstains using a simple DNA extraction kit and capillary electrophoresis.

    PubMed

    Yamamoto, T; Kojima, T; Nozawa, H; Huang, X L; Ohtaki, H; Uchihi, R; Tamaki, K; Katsumata, Y

    1997-10-01

    A rapid typing method for STR analysis from bloodstains was devised. DNA from three-year-old bloodstains of six individuals was extracted with a cationic detergent in a newly-released kit. Amounts of DNA extracted by the kit were 16.2 +/- 1.8 ng from 10 microliters of fresh blood samples. DNA was also extracted from bloodstains of three months and three years left at room temperature, and amounts of extracted DNA were estimated at 2.2 +/- 1.0 ng and 0.1 +/- 0.1 ng, respectively. The PCR mixture supplied with the kit, plus three sets of fluorescently labeled primers, were directly added to the tube used for DNA extraction. The amplified products were then analyzed by capillary electrophoresis, and the obtained data were automatically sized and typed using computer software. Three genotypes of all bloodstains except one sample at a single locus were determined correctly as those of freshly collected blood from the same individuals although some stains contained no more than 0.1 ng. Using this typing system, we were able to type three STR loci, HUMTH01, HUMF13A1 and HUMFES/FPS, from a single bloodstain within five hours. This typing is particularly useful for forensic practice. PMID:9436368

  11. A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.

    PubMed

    Freifeld, Alison G; Simonsen, Kari A; Booth, Christine S; Zhao, Xing; Whitney, Scott E; Karre, Teresa; Iwen, Peter C; Viljoen, Hendrik J

    2012-01-01

    We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases. PMID:22402170

  12. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

    PubMed Central

    2012-01-01

    A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 ?L droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3?min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10?min. Following extraction, the 1500?bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10?min for 30?cycles. The total assay time was 23?min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 ?L droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5?min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation. PMID:22947281

  13. Novel DNA Extraction Method Unveiled the Ancient Hot Deep Biosphere Concealed in Terrestrial Sedimentary Rocks

    NASA Astrophysics Data System (ADS)

    Kouduka, M.; Suko, T.; Okuzawa, K.; Fukuda, A.; Nanba, K.; Yamamoto, M.; Sakata, S.; Ito, K.; Suzuki, Y.

    2009-12-01

    It has been proposed that the hot deep biosphere is distributed deeply in the crust of the Earth. Below the upper limit of life, prokaryotic habitats extend at a depth of 4 km within a typical range of thermal gradients (2.5-3°C/100m). In contrary, large geothermal gradients allow the hot deep biosphere approaching to the Earth’s surface. We conducted aseptic and deoxygenated drilling targeting Miocene marine siliceous rocks in a tectonically stable inland fore-arc basin in central Japan. Although the in-situ groundwater temperature was 30.2°C at a maximum drilling depth of 352 mbgl, opal-CT and clinoptilolite, which commonly form as a result of progressive burial at 30-60°C and over 70°C, respectively, were detected by XRD analysis. However, burial-driven degradation and transformation of sterols (sterene to sterane) was not evident in the core samples. As presented by Y. Suzuki et al. in this meeting, extant microbial populations were dominated by Pseudomonas spp. and Flavobacterium spp. with cell numbers ranging ~107-108 cells/cm3 rock. Despite the abundance of microbial cells, DNA were not extracted from the core samples by conventional methods. We developed a DNA extraction method to avoid binding of DNA onto the siliceous mineral matrix by heating under alkaline conditions, which resulted in the successful retrieval of PCR-amplifiable DNA. Unexpectedly, 16S rRNA gene sequences closely related thermophilic Geobacillus stearothermophilus and Thermus thermophilus were dominant in the clone libraries from 300- and 350-m deep core samples, while those almost identical to the Pseudomonas spp. were minor. Based on the correlation between the GC contents of 16S rRNA gene sequences and growth temperatures of prokaryotes, the estimated growth temperatures were 90.0°C (G+C=66.3%) and 67.5°C (G+C=61.3%) for G. stearothermophilus and T. thermophilus, respectively. From these results, it is implied that temperature rise in the past led to the colonization of thermophilic bacteria and the transformation of silica minerals in the deep subsurface. As intensive erosion is unlikely around the drilling site, a short period of hydrothermal activities rather than long-term burial at great depth caused high temperature conditions, which might explain the lack of maturity in hydrocarbon. A novel DNA-based approach coupled mineralogical and organic geochemical analyses has the potential to reconstruct ancient biogeochemical processes mediated in the deep subsurface as well as geothermal history. This study was supported by grants from the Nuclear and Industrial Safety Agency (NISA) and Japan Nuclear Energy Safety Organization (JNES).

  14. Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

    PubMed Central

    Yusof, Yasmin Anum Mohd; Md. Saad, Suhana; Makpol, Suzana; Shamaan, Nor Aripin; Ngah, Wan Zurinah Wan

    2010-01-01

    OBJECTIVES: The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2. INTRODUCTION: The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti?cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail. METHODS: HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0?4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis. RESULTS: Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro? apoptotic proteins P53, Bax and caspase?3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti?apoptotic protein Bcl?2. CONCLUSIONS: Chlorella vulgaris may have anti?cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase?3 proteins and through a reduction of Bcl?2 protein, which subsequently lead to increased DNA damage and apoptosis. PMID:21340229

  15. DNA DNA DNA (d)DNA DNA DNA

    E-print Network

    Hagiya, Masami

    DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

  16. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences

    PubMed Central

    Wagner Mackenzie, Brett; Waite, David W.; Taylor, Michael W.

    2015-01-01

    The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates. PMID:25741335

  17. Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shotgun lipidomics approach.

    PubMed

    Wang, Miao; Hayakawa, Jun; Yang, Kui; Han, Xianlin

    2014-02-18

    Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well-known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost-effective and high throughput methodologies for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multidimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/?L), high specificity, and broad linear dynamics range (2500-fold) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) versus a 5-fold difference between 3 month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states. PMID:24432906

  18. Characterization and Quantification of Diacylglycerol Species in Biological Extracts after One-step Derivatization: A Shotgun Lipidomics Approach

    PubMed Central

    Wang, Miao; Hayakawa, Jun; Yang, Kui; Han, Xianlin

    2014-01-01

    Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost effective and high throughput methodology for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/µl), high specificity, and broad linear dynamics range (2,500 folds) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) vs. a 5-fold difference between 3-month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states. PMID:24432906

  19. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    PubMed Central

    2010-01-01

    Background A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. Findings We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit. Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. Conclusions We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications. PMID:20840759

  20. Testing compatibility between molecular and morphological techniques for arthropod systematics: a minimally destructive DNA extraction method that preserves morphological integrity, and the effect of lactic acid on DNA quality

    Microsoft Academic Search

    Pierre Paquin; Cor J. Vink

    2009-01-01

    Three practical aspects related to the preservation and destruction of DNA and\\/or morphological characters of spiders were\\u000a examined: potential morphological damage during non-destructive DNA extraction was assessed by counting trichobothria, a fragile\\u000a sensorial feature found on spider legs; the effect on yield of non-destructive DNA extraction; and whether possible DNA degradation\\u000a is caused by residues of lactic acid, which is

  1. Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error.

    PubMed

    Brandfass, Christoph; Karlovsky, Petr

    2008-11-01

    Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5-1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5-1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed. PMID:19330077

  2. Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes

    PubMed Central

    2013-01-01

    Background Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). Methods Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. Results The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. Conclusion Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants. PMID:24279749

  3. Sensing cocaine in saliva with attenuated total reflection infrared (ATR-IR) spectroscopy combined with a one-step extraction method

    NASA Astrophysics Data System (ADS)

    Hans, Kerstin M.-C.; Gianella, Michele; Sigrist, Markus W.

    2012-03-01

    On-site drug tests have gained importance, e.g., for protecting the society from impaired drivers. Since today's drug tests are majorly only positive/negative, there is a great need for a reliable, portable and preferentially quantitative drug test. In the project IrSens we aim to bridge this gap with the development of an optical sensor platform based on infrared spectroscopy and focus on cocaine detection in saliva. We combine a one-step extraction method, a sample drying technique and infrared attenuated total reflection (ATR) spectroscopy. As a first step we have developed an extraction technique that allows us to extract cocaine from saliva to an almost infrared-transparent solvent and to record ATR spectra with a commercially available Fourier Transform-infrared spectrometer. To the best of our knowledge this is the first time that such a simple and easy-to-use one-step extraction method is used to transfer cocaine from saliva into an organic solvent and detect it quantitatively. With this new method we are able to reach a current limit of detection around 10 ?g/ml. This new extraction method could also be applied to waste water monitoring and controlling caffeine content in beverages.

  4. Evaluation of a semi-automated, magnetic bead-based DNA extraction method for genetic fingerprinting of forensic casework samples

    Microsoft Academic Search

    Barbara Haak; Andrea Porsche; Kai Vollack; Peter Zimmermann; Werner Pflug

    2008-01-01

    In order to cope with the demanding workload for DNA profiling of forensic casework samples a concept for a semi-automated processing system was developed at the Landeskriminalamt (Office of Criminal Investigation) Baden-Württemberg, Germany [K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, this issue]. The applied magnetic bead extraction method is based

  5. A rapid method for extraction of cotton ( Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

    Microsoft Academic Search

    Andrew H. Paterson; Curt L. Brubaker; Jonathan F. Wendel

    1993-01-01

    Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances.\\u000a We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP\\u000a and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol\\u000a oxidase inhibitors

  6. Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

    Microsoft Academic Search

    Kim Steegen; Els Demecheleer; Kenny Dauwe; Kishor Mandaliya; Walter Jaoko; Jean Plum; Marleen Temmerman; Chris Verhofstede

    2007-01-01

    Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naive patients. Amplification and sequencing of RNA and DNA was performed

  7. Ethanolic extract of the Goldenseal, Hydrastis canadensis, has demonstrable chemopreventive effects on HeLa cells in vitro: Drug-DNA interaction with calf thymus DNA as target.

    PubMed

    Saha, Santu Kumar; Sikdar, Sourav; Mukherjee, Avinaba; Bhadra, Kakali; Boujedaini, Naoual; Khuda-Bukhsh, Anisur Rahman

    2013-07-01

    This study tested chemotherapeutic potential of Hydrastis canadensis (HC) extract in HeLa cells in vitro, with emphasis on its drug-DNA interaction and apoptosis induction ability. Nuclear uptake of HC by DAPI, Ao/Eb staining and internucleosomal DNA damage by comet assay was studied through fluorescence microscopy. Possible changes in MMP and apoptotic signalling events were critically analyzed. Cell cycle progression studied through FACS and fragmented DNA through "TUNEL" assay were critically analyzed. RT-PCR studies were conducted for analyzing Cyt-C and Bax translocation in mitochondrial and cytosolic extracts, and Caspase 3 in whole cell lysate. Role of p53-mediated regulation of NF-?? and TNF-? was elucidated by Western blot analysis. Data of CD and Tm profile of CT-DNA were analyzed. Overall results indicated anti-cancer potential of HC through its ability to induce apoptosis, and interaction with CT-DNA that changed structural conformation of DNA, proving HC to be a promising candidate for chemoprevention. PMID:23628949

  8. Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

    Microsoft Academic Search

    J. Kozdrój; J. D. van Elsas

    2000-01-01

    We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns\\u000a obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods,\\u000a i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction\\u000a followed by DNA extraction, and

  9. Low ionic strength extraction of nuclease-treated nuclei destroys the attachment of transcriptionally active DNA to the nuclear skeleton.

    PubMed Central

    Razin, S V; Yarovaya, O V; Georgiev, G P

    1985-01-01

    We have studied how the conditions in which the nuclear matrix is isolated influence the association of transcribing DNA with the nuclear matrix. Extraction of nuclease-treated nuclei with a low ionic strength solution before a high salt nuclei with a low ionic strength solution before a high salt extraction completely abolishes this association. However, RNA removal by RNAase treatment does not affect the binding of transcribing DNA to the nuclear matrix. The nature of the association of active genes with the nuclear matrix is discussed. Images PMID:2414739

  10. Efficient extraction of virus DNA by NucliSens Extractor allows sensitive detection of hepatitis B virus by PCR.

    PubMed

    Gobbers, E; Oosterlaken, T A; van Bussel, M J; Melsert, R; Kroes, A C; Claas, E C

    2001-12-01

    The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification- and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80 degrees C (10 min) and at 37 degrees C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes. PMID:11724842

  11. Efficient Extraction of Virus DNA by NucliSens Extractor Allows Sensitive Detection of Hepatitis B Virus by PCR

    PubMed Central

    Gobbers, Erik; Oosterlaken, Tom A. M.; van Bussel, Mario J. A. W. M.; Melsert, Roel; Kroes, Aloys C. M.; Claas, Eric C. J.

    2001-01-01

    The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification- and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80°C (10 min) and at 37°C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes. PMID:11724842

  12. High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters, Determined Using a Modified DNA Extraction Method? †

    PubMed Central

    Urakawa, Hidetoshi; Martens-Habbena, Willm; Stahl, David A.

    2010-01-01

    Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community. PMID:20118363

  13. One-Step Analysis of DNA/Chitosan Complexes by Field-Flow Fractionation Reveals Particle Size and Free Chitosan Content

    E-print Network

    Buschmann, Michael

    One-Step Analysis of DNA/Chitosan Complexes by Field-Flow Fractionation Reveals Particle Size with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV particles.2 Asym- metrical flow field-flow fractionation (AF4), an analytical separation technique in which

  14. Ion-Channel Genosensor for the Detection of Specific DNA Sequences Derived from Plum Pox Virus in Plant Extracts

    PubMed Central

    Malecka, Kamila; Michalczuk, Lech; Radecka, Hanna; Radecki, Jerzy

    2014-01-01

    A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3?/4? as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1–8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10–50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal. PMID:25302809

  15. What do results of common sequential fractionation and single-step extractions tell us about P binding with Fe and Al compounds in non-calcareous sediments?

    PubMed

    Jan, Ji?í; Borovec, Jakub; Kopá?ek, Ji?í; Hejzlar, Josef

    2013-02-01

    Correct identification of P forms together with their main Fe and Al binding partners in non-calcareous sediments is of crucial importance for evaluation of P cycling in water bodies. In this paper, we assess extraction methods frequently used for this purpose, i.e., a sequential five-step fractionation (water, bicarbonate buffered dithionite solution (BD), NaOH, HCl, nitric-perchloric acid), ascorbate extraction (pH ~7.5), and oxalate extraction (pH ~3), directly on a range of laboratory prepared Fe and Al minerals enriched with adsorbed P. Extraction selectivity and efficiency for particular P, Fe and Al forms were also verified by specific combinations of these extraction methods applied on freshwater sediment samples. In the sequential fractionation, BD was highly effective in dissolving both amorphous and crystalline Fe (hydr)oxides and the associated P, while neither FeS nor Al (hydr)oxides were dissolved. The following NaOH extraction effectively dissolved both amorphous and crystalline Al (hydr)oxides. The high solubilizing power of BD and NaOH to dissolve crystalline Fe and Al oxides that have only a small P-sorption ability prevents the use of resulting Fe/P and Al/P ratios as simple predictors of total P sorption capacity of sediments and soils. Ascorbate non-selectively extracted small proportions of FeS and amorphous Fe and Al (hydr)oxides, but significant amounts of adsorbed P, which hinders its use for the characterization of P forms in non-calcareous sediments. Similar nonselective characteristics were found for oxalate extractions. As oxalate extracts most of the adsorbed phosphate, it is not possible to use it unambiguously to determine specific Fe/P and Al/P ratios of active complexes. However, this method is convenient (and more selective than NaOH step in the sequential fractionation) for the determination of amorphous Al (hydr)oxides. PMID:23218245

  16. Molecular Dynamics Simulations of the 136 Unique Tetranucleotide Sequences of DNA Oligonucleotides. II: Sequence Context Effects on the Dynamical Structures of the 10 Unique Dinucleotide Steps

    PubMed Central

    Dixit, Surjit B.; Beveridge, David L.; Case, David A.; Cheatham, Thomas E.; Giudice, Emmanuel; Lankas, Filip; Lavery, Richard; Maddocks, John H.; Osman, Roman; Sklenar, Heinz; Thayer, Kelly M.; Varnai, Péter

    2005-01-01

    Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA. Calculations were carried out on the 136 cases embedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All simulations were carried out using a well-defined state-of-the-art MD protocol, the AMBER suite of programs, and the parm94 force field. In a previous article (Beveridge et al. 2004. Biophysical Journal. 87:3799–3813), the research design, details of the simulation protocol, and informatics issues were described. Preliminary results from 15 ns MD trajectories were presented for the d(CpG) step in all 10 unique sequence contexts. The results indicated the sequence context effects to be small for this step, but revealed that MD on DNA at this length of trajectory is subject to surprisingly persistent cooperative transitions of the sugar-phosphate backbone torsion angles ? and ?. In this article, we report detailed analysis of the entire trajectory database and occurrence of various conformational substates and its impact on studies of context effects. The analysis reveals a possible direct correspondence between the sequence-dependent dynamical tendencies of DNA structure and the tendency to undergo transitions that “trap” them in nonstandard conformational substates. The difference in mean of the observed basepair step helicoidal parameter distribution with different flanking sequence sometimes differs by as much as one standard deviation, indicating that the extent of sequence effects could be significant. The observations reveal that the impact of a flexible dinucleotide such as CpG could extend beyond the immediate basepair neighbors. The results in general provide new insight into MD on DNA and the sequence-dependent dynamical structural characteristics of DNA. PMID:16169978

  17. Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure

    PubMed Central

    Terrat, Sébastien; Christen, Richard; Dequiedt, Samuel; Lelièvre, Mélanie; Nowak, Virginie; Regnier, Tiffanie; Bachar, Dipankar; Plassart, Pierre; Wincker, Patrick; Jolivet, Claudy; Bispo, Antonio; Lemanceau, Philippe; Maron, Pierre?Alain; Mougel, Christophe; Ranjard, Lionel

    2012-01-01

    Summary Three soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15?000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies. PMID:21989224

  18. Extraction of transcription regulatory signals from genome-wide DNA–protein interaction data

    PubMed Central

    Garten, Yael; Kaplan, Shai; Pilpel, Yitzhak

    2005-01-01

    Deciphering gene regulatory network architecture amounts to the identification of the regulators, conditions in which they act, genes they regulate, cis-acting motifs they bind, expression profiles they dictate and more complex relationships between alternative regulatory partnerships and alternative regulatory motifs that give rise to sub-modalities of expression profiles. The ‘location data’ in yeast is a comprehensive resource that provides transcription factor–DNA interaction information in vivo. Here, we provide two contributions: first, we developed means to assess the extent of noise in the location data, and consequently for extracting signals from it. Second, we couple signal extraction with better characterization of the genetic network architecture. We apply two methods for the detection of combinatorial associations between transcription factors (TFs), the integration of which provides a global map of combinatorial regulatory interactions. We discover the capacity of regulatory motifs and TF partnerships to dictate fine-tuned expression patterns of subsets of genes, which are clearly distinct from those displayed by most genes assigned to the same TF. Our findings provide carefully prioritized, high-quality assignments between regulators and regulated genes and as such should prove useful for experimental and computational biologists alike. PMID:15684410

  19. The optimization of essential oils supercritical CO2 extraction from Lavandula hybrida through static-dynamic steps procedure and semi-continuous technique using response surface method

    PubMed Central

    Kamali, Hossein; Aminimoghadamfarouj, Noushin; Golmakani, Ebrahim; Nematollahi, Alireza

    2015-01-01

    Aim: The aim of this study was to examine and evaluate crucial variables in essential oils extraction process from Lavandula hybrida through static-dynamic and semi-continuous techniques using response surface method. Materials and Methods: Essential oil components were extracted from Lavandula hybrida (Lavandin) flowers using supercritical carbon dioxide via static-dynamic steps (SDS) procedure, and semi-continuous (SC) technique. Results: Using response surface method the optimum extraction yield (4.768%) was obtained via SDS at 108.7 bar, 48.5°C, 120 min (static: 8×15), 24 min (dynamic: 8×3 min) in contrast to the 4.620% extraction yield for the SC at 111.6 bar, 49.2°C, 14 min (static), 121.1 min (dynamic). Conclusion: The results indicated that a substantial reduction (81.56%) solvent usage (kg CO2/g oil) is observed in the SDS method versus the conventional SC method. PMID:25598636

  20. Detection of Transient Bacteraemia following Dental Extractions by 16S rDNA Pyrosequencing: A Pilot Study

    PubMed Central

    Benítez-Páez, Alfonso; Álvarez, Maximiliano; Belda-Ferre, Pedro; Rubido, Susana; Mira, Alex; Tomás, Inmaculada

    2013-01-01

    Objective The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing. Methods The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene. Results Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.4±1.7 bacterial families and 22.8±1.1 genera per sample. Conclusion The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied. PMID:23469240

  1. Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

    Microsoft Academic Search

    U. Morris; B. Aydin-Schmidt; D. Shakely; A. Martensson; L. Jornhagen; A. S. Ali; M. I. Msellem; M. Petzold; J. P. Gil; P. Ferreira; A. Bjorkman

    2013-01-01

    BACKGROUND: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and

  2. Evaluation of DNA extraction methods from mouse stomachs for the quantification of H. pylori by real-time PCR

    Microsoft Academic Search

    Yvonne Roussel; Mark Wilks; Andrew Harris; Charles Mein; Soad Tabaqchali

    2005-01-01

    Real-time PCR methods have recently been developed for the quantification of Helicobacter pylori from infected mouse stomachs. However, the extent to which results is affected by the efficiency of different methods of DNA extraction and the degree of inhibition of the subsequent PCR have largely been ignored. In this study, mouse stomachs were processed using two homogenisation methods: complete disruption

  3. Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods

    Microsoft Academic Search

    J. L. Gonzales; A. Loza; E. Chacon

    2006-01-01

    There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers

  4. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis

    Microsoft Academic Search

    Richard A. Haugland; Nichole Brinkman; Stephen J. Vesper

    2002-01-01

    Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan™) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of

  5. Variation of RAPD-fingerprint patterns using different DNA-extraction methods with Gram-positive bacteria

    Microsoft Academic Search

    Y. Abed; A. Davin; R. N. Charrel; C. Bollet; P. Micco

    1995-01-01

    The effect of the DNA-extraction method used on fingerprint patterns of RAPD was studied usingStaphylococcus epidermidis andStreptococcus faecalis. The three methods tested (Chelex, microwave and phenol\\/chloroform) led to significantly different RAPD patterns. The microwave technique generated reproducible patterns and seems the most suitable for RAPD analysis of Gram-positive bacteria.

  6. Evaluation of Antioxidant and DNA Damage Protection Activity of the Hydroalcoholic Extract of Desmostachya bipinnata L. Stapf

    PubMed Central

    Bhimathati, Solomon Sunder Raj

    2014-01-01

    Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18 ± 3.47??g/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50??g/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases. PMID:24574873

  7. Protective effect of Kappaphycus alvarezii (Rhodophyta) extract against DNA damage induced by mercury chloride in marine fish

    Microsoft Academic Search

    N. Nagarani; V. JanakiDevi; M. YokeshBabu; A. K. Kumaraguru

    2012-01-01

    Marine organisms are continuously exposed to agents, both exogenous and endogenous, that damage DNA. Consequently, it is important to determine the ability of compounds to provide protection against damaging chemicals. The aim of this study was to evaluate the anti-genotoxic activity of crude aqueous extracts of Kappaphycus alvarezii (Rhodophyceae), collected from the Southeast coast of India. This study focused on

  8. Mild two-step method to construct DNA-conjugated silicon nanoparticles: scaffolds for the detection of microRNA-21.

    PubMed

    Su, Xiaoye; Kuang, Li; Battle, Cooper; Shaner, Ted; Mitchell, Brian S; Fink, Mark J; Jayawickramarajah, Janarthanan

    2014-10-15

    We describe a novel two-step method, starting from bulk silicon wafers, to construct DNA conjugated silicon nanoparticles (SiNPs). This method first utilizes reactive high-energy ball milling (RHEBM) to obtain alkene grafted SiNPs. The alkene moieties are subsequently reacted with commercially available thiol-functionalized DNA via thiol-ene click chemistry to produce SiNP DNA conjugates wherein the DNA is attached through a covalent thioether bond. Further, to show the utility of this synthetic strategy, we illustrate how these SiNP ODN conjugates can detect cancer-associated miR-21 via a fluorescence ON strategy. Given that an array of biological molecules can be prepared with thiol termini and that SiNPs are biocompatible and biodegradable, we envision that this synthetic protocol will find utility in salient SiNP systems for potential therapeutic and diagnostic applications. PMID:25243490

  9. Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples.

    PubMed

    Dauphin, Leslie A; Moser, Benjamin D; Bowen, Michael D

    2009-01-01

    This study evaluated five commercial extraction kits for their ability to recover DNA from Bacillus anthracis spores and spiked environmental samples. The kits evaluated represent the major types of methodologies which are commercially available for DNA or total nucleic acid extraction, and included the ChargeSwitch gDNA Mini Bacteria Kit, NucliSens Isolation Kit, Puregene Genomic DNA Purification Kit, QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. Extraction methods were performed using the spores of eight virulent strains of B. anthracis. Viability testing of nucleic acid extracts showed that the UltraClean kit was the most efficient at depleting samples of live B. anthracis spores. TaqMan real-time PCR analysis revealed that the NucliSens, QIAamp and UltraClean kits yielded the best level of detection from spore suspensions. Comparisons of processed samples from spiked swabs and three powder types indicated that DNA extraction using the UltraClean kit resulted in the most consistently positive results and the lowest limit of detection. This study demonstrated that different nucleic extraction methodologies, represented here by various commercial extraction kits, differ in their ability to inactivate live B. anthracis spores as well as DNA yield and purity. In addition, the extraction method used can influence the sensitivity of real-time PCR assays for B. anthracis. PMID:18824041

  10. Do DNA extraction methods and Taq polimerase quality improve the double repetitive element (DRE) PCR typing method for Mycobacterium tuberculosis strains?

    Microsoft Academic Search

    Hebe Rodrigues Cavalcanti; Elizabeth Marques; Leila de Souza Fonseca; Maria Helena Féres Saad

    2007-01-01

    Double repetitive element (DRE) PCR amplification is a simple Mycobacterium tuberculosis typing method, however amplification failure or poor resolution of bands commit its efficacy. In order to verify if whether or not these features could be minimized by improving DNA extraction procedures or Taq polymerise quality, DRE-PCR was performed on 24 M. tuberculosis DNA samples extracted by heat-shock, mechanical and

  11. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  12. Protective effect of water yam (Dioscorea alata L.) extract on the copper-driven fenton reaction and X-ray induced DNA damage in vitro.

    PubMed

    Wang, Tsu-Shing; Liang, Shih-Jyue; Lii, Chong-Kuei; Liu, Sin-Yie

    2004-04-01

    The rhizome extract of Dioscorea has been shown to possess radical scavenging activity. In this study, the protective effect of water yam (Dioscorea alata L.) rhizome extract on calf thymus DNA and plasmid DNA strand breakage by the copper-driven Fenton reaction and X-irradiation was examined. The protective activity in vitro of four lyophilized extracts obtained from yam rhizomes: (1) aqueous extract (YAE); (2) 30% ethanolic extract (YEE); (3) aqueous extract boiled for 30 min (BYAE); and (4) 30% ethanolic extract boiled for 30 min (BYEE) were evaluated by ethidium bromide binding assay and DNA nicking assay. The YAE, YEE, and BYEE effectively inhibited the copper-driven Fenton reaction-induced damage of calf thymus DNA, while inhibition was less pronounced in the case of X-ray induced strand breakage of plasmid DNA. While BYAE potently inhibited X-ray induced strand breaks in plasmid pGL3 DNA, it failed to inhibit, and even greatly enhanced copper-H(2)O(2) induced damage of calf thymus DNA. The present results demonstrate strong copper chelating and weak hydroxyl radical scavenging activities in yam rhizome extracts, and these activities may vary depending on the procedures used in preparing the extract. PMID:15162369

  13. A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga

    PubMed Central

    Greco, Maria; Sáez, Claudio A.; Brown, Murray T.; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10–11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg?1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae. PMID:24867404

  14. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

    PubMed

    Greco, Maria; Sáez, Claudio A; Brown, Murray T; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1) fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae. PMID:24867404

  15. Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis

    PubMed Central

    2014-01-01

    Background In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies. Results In this study we conducted a systematic comparison of the DNA extraction methods used by the two major collaborative efforts: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol. Conclusions Whereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies. PMID:24949196

  16. Rapid detection of Rhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

    Microsoft Academic Search

    Masaru Matsumoto; Naruto Furuya; Yoichi Takanami; Nobuaki Matsuyama

    1997-01-01

    Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR)\\u000a was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract\\u000a parasite DNA from diseased

  17. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    Microsoft Academic Search

    Randy P. Revetta; Robin S. Matlib

    2011-01-01

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA\\u000a extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic\\u000a analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly\\u000a 62% of all DNA sequences analyzed.

  18. Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC\\/MS

    Microsoft Academic Search

    W. Weinmann; M. Renz; S. Vogt; S. Pollak

    2000-01-01

    A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the\\u000a simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure\\u000a are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA.\\u000a SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark

  19. Application of a modified BCR sequential extraction (three-step) procedure for the determination of extractable trace metal contents in a sewage sludge amended soil reference material (CRM 483), complemented by a three-year stability study of acetic acid and EDTA extractable metal content.

    PubMed

    Rauret, G; López-Sánchez, J F; Sahuquillo, A; Barahona, E; Lachica, M; Ure, A M; Davidson, C M; Gomez, A; Lück, D; Bacon, J; Yli-Halla, M; Muntau, H; Quevauviller, P

    2000-06-01

    This paper provides additional data on a sewage sludge amended soil certified reference material, CRM 483, which was certified in 1997 for its EDTA and acetic acid extractable contents of some trace metals, following standardised extraction procedures. The additional work aimed to test the long-term stability of the material and the applicability of an improved version of the BCR three-step sequential extraction procedure on the sewage sludge amended soil (CRM 483). The paper demonstrates the CRM 483 long-term stability for EDTA and acetic acid extractable contents of Cd, Cr, Cu, Ni, Pb and Zn and gives the results (obtained in the framework of an interlaboratory study) for the extractable contents of the same elements in the CRM 483, following the BCR three-step sequential extraction scheme. The aqua regia extractable contents following the ISO 11466 Standard are also given. The data are given as indicative (not certified) values. PMID:11256704

  20. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    PubMed

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100?l of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants. PMID:25681524

  1. COMPARISON OF DNA EXTRACTION METHODS FOR ANALYSIS OF TURKEY CECAL MICROBIOTA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To compare fourteen DNA isolation protocols from eight commercially available kits, turkey cecal microbial diversity was evaluated. Kits included Qbiogene Fast DNA, Qiagen QIAamp DNA Stool, MoBio Ultra Clean Fecal, MoBio Ultra Clean Soil, Roche High Pure, Epicentre Soilmaster, BioRad Aquapure DNA a...

  2. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

    PubMed Central

    Zelenaya-Troitskaya, O; Perlman, P S; Butow, R A

    1995-01-01

    The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA. Images PMID:7621838

  3. Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid-liquid extraction followed by high performance liquid chromatography.

    PubMed

    Valdivielso, Izaskun; Bustamante, María Ángeles; Ruiz de Gordoa, Juan Carlos; Nájera, Ana Isabel; de Renobales, Mertxe; Barron, Luis Javier R

    2015-04-15

    Carotenoids and tocopherols from botanical species abundant in Atlantic mountain grasslands were simultaneously extracted using one-step solid-liquid phase. A single n-hexane/2-propanol extract containing both types of compounds was injected twice under two different sets of HPLC conditions to separate the tocopherols by normal-phase chromatography and carotenoids by reverse-phase mode. The method allowed reproducible quantification in plant samples of very low amounts of ?-, ?-, ?- and ?-tocopherols (LOD from 0.0379 to 0.0720 ?g g(-1) DM) and over 15 different xanthophylls and carotene isomers. The simplified one-step extraction without saponification significantly increased the recovery of tocopherols and carotenoids, thereby enabling the determination of ?-tocopherol acetate in plant samples. The two different sets of chromatographic analysis provided near baseline separation of individual compounds without interference from other lipid compounds extracted from plants, and a very sensitive and accurate detection of tocopherols and carotenoids. The detection of minor individual components in botanical species from grasslands is nowadays of high interest in searching for biomarkers for foods derived from grazing animals. PMID:25466080

  4. Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

    PubMed Central

    Sugasawa, K; Murakami, Y; Miyamoto, N; Hanaoka, F; Ui, M

    1990-01-01

    A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure. Images PMID:2168970

  5. Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues

    PubMed Central

    Waggoner, Jesse J.

    2014-01-01

    Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types. PMID:24765569

  6. Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues.

    PubMed

    Waggoner, Jesse J; Pinsky, Benjamin A

    2014-01-01

    Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types. PMID:24765569

  7. Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

    PubMed

    Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J

    2013-01-01

    The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of ?1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice. PMID:22836616

  8. Rapid Detection of Pneumocystis carinii in Bronchoalveolar Lavage Specimens from Human Immunodeficiency Virus Infected Patients: Use of a Simple DNA Extraction Procedure and Nested PCR

    Microsoft Academic Search

    MEJA RABODONIRINA; DIDIER RAFFENOT; LAURENT COTTE; MARTINE MAYENCON; GERMAINE BAYLE; FLORENCE PERSAT; FABIEN RABATEL; CHRISTIAN TREPO; DOMINIQUE PEYRAMOND; MARIE-ANTOINETTE PIENS

    1997-01-01

    We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed

  9. Kinetic Parameter Extraction of Square Wave Voltammograms from DNA-Modified Gold Electrodes

    NASA Astrophysics Data System (ADS)

    McWilliams, Marc; Wohlgamuth, Chris; Slinker, Jason

    2012-10-01

    The field of surface bound electrochemistry is important in a variety of applications specifically sensing. A fundamental understanding of the processes involved could help to improve detection limits, optimize rates of detection and direct changes in device design. Accurate extraction of electrochemical kinetic parameters such as the rate constant k and charge transfer coefficient ? from cyclic voltammograms can be challenging when confronted with large background currents and relatively weak signals. The commonly used technique of Laviron analysis is both time consuming and somewhat subjective. Square wave voltammetry (SWV) is therefore an ideal alternative method given that it maximizes signal while minimizing capacitive effects. In this experiment kinetic parameters of DNA-modified gold electrodes are obtained from SWV curves through background subtraction followed by nonlinear least squares fitting using a first order quasi-reversible surface process model. The fitting is accomplished using the Nelder-Mead simplex algorithm with standard parameters and a convergence condition of less than 0.0001%. General agreement with experimental data is shown with varying levels of confidence. Difficulties specific to this experiment are discussed as well as the possible benefits of utilizing the Bayesian statistical approach of nested sampling when confronted with multiple peaks of interest and the background source is well defined.

  10. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  11. An alternative method for DNA extraction and PCR identification of Entamoeba histolytica and E. dispar in fecal samples.

    PubMed

    Vianna, E N; Costa, J O; Santos, C K S; Cury, M C; Silva, E F; Costa, A O; Gomes, M A

    2009-06-01

    Since it is known that Entamoeba dispar is non-pathogenic and morphologically similar to E. histolytica, there are many targets used in PCR for differentiating these species. However, obtaining high quality DNA from fecal samples is fundamental for PCR. Most methods are laborious or use kits that make diagnosis expensive. In the present work, a new simple, fast and cheap technique of DNA extraction from fecal samples was combined with a PCR for an episomal target in order to identify E. histolytica and E. dispar in feces. PMID:19486545

  12. Uncaria tomentosa extracts protect human erythrocyte catalase against damage induced by 2,4-D-Na and its metabolites.

    PubMed

    Bukowska, Bo?ena; Bors, Milena; Gulewicz, Krzysztof; Koter-Michalak, Maria

    2012-06-01

    The effect of ethanolic and aqueous extracts from leaves and bark of Uncaria tomentosa was studied, with particular attention to catalase activity (CAT - EC. 1.11.1.6). We observed that all tested extracts, at a concentration of 250 ?g/mL were not toxic to erythrocyte catalase because they did not decreased its activity. Additionally, we investigated the protective effect of extracts on changes in CAT activity in the erythrocytes incubated with sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D-Na) and its metabolites i.e., 2,4-dichlorophenol (2,4-DCP) and catechol. Previous investigations showed that these chemicals decreased activity of erythrocyte catalase (Bukowska et al., 2000; Bukowska and Kowalska, 2004). The erythrocytes were divided into two portions. The first portion was incubated for 1 and 5h at 37°C with 2,4-D-Na, 2,4-DCP and catechol, and second portion was preincubated with extracts for 10 min and then incubated with xenobiotics for 1 and 5h. CAT activity was measured in the first and second portion of the erythrocytes. We found a protective effect of the extracts from U. tomentosa on the activity of catalase incubated with xenobiotics studied. Probably, phenolic compounds contained in U. tomentosa scavenged free radicals, and therefore protected active center (containing -SH groups) of catalase. PMID:22426356

  13. Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

    PubMed Central

    Rabodonirina, M; Raffenot, D; Cotte, L; Boibieux, A; Mayençon, M; Bayle, G; Persat, F; Rabatel, F; Trepo, C; Peyramond, D; Piens, M A

    1997-01-01

    We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens. PMID:9350726

  14. Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: The inhibitory effect of linoleic acid on the DNA-binding step

    SciTech Connect

    Jung, Kyung Chae [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Rhee, Ho Sung [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Park, Chi Hoon [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Yang, Chul-Hak [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of)]. E-mail: chulyang@plaza.snu.ac.kr

    2005-08-19

    c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90({+-}0.54) x 10{sup -7} M for Max85/Max85 homodimer, 6.85({+-}0.25) x 10{sup -3} M for Myc87/Myc87 homodimer, and 2.55({+-}0.29) x 10{sup -8} M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33({+-}0.21) x 10{sup -9} M for Max85/Max85/DNA, 2.27({+-}0.08) x 10{sup -12} M for Myc87/Myc87/DNA, and 4.43({+-}0.37) x 10{sup -10} M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.

  15. Analysis of plasticizers in poly(vinyl chloride) medical devices for infusion and artificial nutrition: comparison and optimization of the extraction procedures, a pre-migration test step.

    PubMed

    Bernard, Lise; Cueff, Régis; Bourdeaux, Daniel; Breysse, Colette; Sautou, Valérie

    2015-02-01

    Medical devices (MDs) for infusion and enteral and parenteral nutrition are essentially made of plasticized polyvinyl chloride (PVC). The first step in assessing patient exposure to these plasticizers, as well as ensuring that the MDs are free from di(2-ethylhexyl) phthalate (DEHP), consists of identifying and quantifying the plasticizers present and, consequently, determining which ones are likely to migrate into the patient's body. We compared three different extraction methods using 0.1 g of plasticized PVC: Soxhlet extraction in diethyl ether and ethyl acetate, polymer dissolution, and room temperature extraction in different solvents. It was found that simple room temperature chloroform extraction under optimized conditions (30 min, 50 mL) gave the best separation of plasticizers from the PVC matrix, with extraction yields ranging from 92 to 100 % for all plasticizers. This result was confirmed by supplemented Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and gravimetric analyses. The technique was used on eight marketed medical devices and showed that they contained different amounts of plasticizers, ranging from 25 to 36 % of the PVC weight. These yields, associated with the individual physicochemical properties of each plasticizer, highlight the need for further migration studies. PMID:25577357

  16. Speciation and determination of bioavailable arsenic species in soil samples by one-step solvent extraction and high-performance liquid chromatography with inductively coupled plasma mass spectrometry.

    PubMed

    Sun, Jing; Ma, Li; Yang, Zhaoguang; Lee, Hsiaowan; Wang, Lin

    2015-03-01

    A new analytical method was developed to determine the bioavailable arsenic species (arsenite, arsenate, monomethylarsonic acid, and dimethylarsonic acid) in soil samples using high-performance liquid chromatography with inductively coupled plasma mass spectrometry. Bioavailable arsenic was extracted with ammonium phosphate buffer by a simplified one-step solvent extraction procedure. To estimate the effect of variables on arsenic extraction, a two-level Plackett-Burman factorial design was conducted to screen the significant factors that were further investigated by a separate univariate approach. The optimum conditions were confirmed by compromising the stability of arsenic species and the extraction efficiency. The concentration of arsenic species was determined in method blank and soil-certified reference materials both spiked with standard solutions of arsenic species. All the target arsenic species were stable during the whole extraction procedure. Furthermore, the proposed method was applied to release bioavailable arsenic from contaminated soil samples, showing that the major arsenic species in soil samples were inorganic arsenic: arsenite and arsenate, of which the latter was dominant. PMID:25594186

  17. Investigations on hydrolytic activities from Stachybotrys microspora and their use as an alternative in yeast DNA extraction.

    PubMed

    Abdeljalil, Salma; Ben Hmad, Ines; Saibi, Walid; Amouri, Bahia; Maalej, Wiem; Kaaniche, Marwa; Koubaa, Aida; Gargouri, Ali

    2014-02-01

    Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, ?-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. ?-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, ?-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA. PMID:24241970

  18. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  19. A one-step extraction/clean-up method for determination of PCBs, PBDEs and HBCDs in environmental solid matrices.

    PubMed

    Abdallah, Mohamed Abou-Elwafa; Drage, Daniel; Harrad, Stuart

    2013-12-01

    A selective pressurized liquid extraction (S-PLE) method was developed for rapid determination of 3 classes of halogenated organic contaminants in indoor dust, soil and sediment samples. The optimised method used 3?:?2 v/v n-hexane-dichloromethane for extraction of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs). Extraction was performed at 90 °C for 5 min followed by 4 min static time under 1500 psi. Good recoveries of target analytes were obtained after 3 extraction cycles. In-cell cleanup was performed using 10 g of 44% H2SO4 acid silica and 5 g of florisil (secondary fat retainer), while copper powder was used to remove elemental sulfur. The method was validated using NIST SRM2585 and SRM 1941b in addition to an in-house previously characterised soil sample. Measured concentrations of target compounds showed good agreement with the certified values with RSD < 20% indicating the good accuracy and precision of the S-PLE method. Clean extracts provided low noise levels resulting in low method detection limits (<0.03 ng g(-1)) and LOQs (<0.1 ng g(-1)). The method developed was applied successfully to real environmental samples and it provided various advantages over traditional methods including reduced solvent consumption and analysis time, minimal sample contamination and high sample throughput which can be beneficial for environmental monitoring programs dealing with large numbers of samples. PMID:24145825

  20. Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water

    Microsoft Academic Search

    Xin Yu; Michele I. Van Dyke; Andrea Portt; Peter M. Huck

    2009-01-01

    Giardia lamblia is one of the most recognized waterborne protozoan parasites causing gastrointestinal disease. A simple but effective DNA\\u000a extraction protocol for real-time PCR detection from surface water samples was developed in this study. Eleven protocols were\\u000a compared, which consisted of freeze–thaw treatments (liquid N2 and boiling water) and purification using the Qiagen DNeasy kit, together with different combinations of

  1. Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs

    Microsoft Academic Search

    Javier Montero-Pau; Africa Gómez; Joaquín Muñoz

    We describe the application of a simple, low-cost, and effective method of DNA extraction (hot sodium hydroxide and Tris, HotSHOT) to the diapausing propagules of continental aquatic invertebrates for its use in PCR amplification. We illustrate the use of the technique in cladocerans, rotifers, anostracans, notostracans, and copepod diapausing eggs. We compare the performance of the HotSHOT technique to the

  2. Trapping and extraction of target DNA molecules using periodically crossed electrophoresis in the micropillar array

    Microsoft Academic Search

    Soyeon Yi; Kyoung-Sun Seo; Young-Ho Cho

    2003-01-01

    This paper presents a DNA (Deoxyribose Nucleic Acid) extractor based on an electrophoresis using periodically crossed electric fields in a micropillar array. The DNA extractor, having nanometer entropic barriers and micropillar array, was fabricated by micromachining processes. Under the crossed electric field, the DNA molecules, whose reorientation time is longer than the period of the crossed field, are trapped in

  3. DNA damage of mammalian cells by the beef extract mutagen 2-amino-3-methylimidazo[4,5-f]quinoline.

    PubMed

    Caderni, G; Kreamer, B L; Dolara, P

    1983-10-01

    The beef-extract mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was shown, by alkaline elution procedures, to induce DNA damage in radiation-induced mouse leukaemia cells. The effect, which was dose related, occurred in incubations containing S-9 mix derived from polychlorinated biphenyl-induced rat liver but not in the absence of this metabolic activation system. An increased alkaline elution of DNA was also observed following IQ addition to cultures of hepatocytes from 3,3',4,4'-tetrachloroazobenzene-induced rat liver, and the DNA damage was again dose related. IQ has thus been shown to be genotoxic to mammalian cells in the presence of an effective activation system. PMID:6686192

  4. One step green synthesis of silver nano/microparticles using extracts of Trachyspermum ammi and Papaver somniferum.

    PubMed

    Vijayaraghavan, K; Nalini, S P Kamala; Prakash, N Udaya; Madhankumar, D

    2012-06-01

    A novel biosynthesis route for silver nanoparticles (Ag-NPs) was attempted in this present investigation using aqueous extracts of Trachyspermum ammi and Papaver somniferum. The main constituents in T. ammi are thymol, p-cymene and ?-terpinene, while P. somniferum consists of morphine and codeine. The essential oil in T. ammi was found to be a good reducing agent than the alkaloids present in P. somniferum for the formation of biocompatible Ag-NPs. The effectiveness of both the extracts was investigated by using same dosage of extract in the synthesis of silver nanoparticle. The results showed that for the same dosage of extracts the T. ammi synthesized various size triangular shaped nanoparticles measuring from 87 nm, to a fewer nanoparticles having a size of 998 nm diagonally. P. somniferum resulted in almost spherical shaped particle ranging in size between 3.2 and 7.6 ?m diagonally. Future research based on synthesis of size specific nanoparticle based on the optimization of reaction condition would be an interesting area. PMID:22348989

  5. Micro-Doppler Effect Analysis and Feature Extraction in ISAR Imaging With Stepped-Frequency Chirp Signals

    E-print Network

    Luo, Ying

    The micro-Doppler (m-D) effect induced by the rotating parts or vibrations of the target provides a new approach for target recognition. To obtain high range resolution for the extraction of the fine m-D signatures of an ...

  6. Validating DNA barcodes: A non-destructive extraction protocol enables simultaneous vouchering of DNA and morphological vouchers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Morphology-based keys support accurate identification of many taxa. However, identification can be difficult for taxa that are not well studied, very small, members of cryptic species complexes, or represented by immature stages. For such cases, DNA barcodes may provide diagnostic characters. Ecolog...

  7. Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system.

    PubMed

    Kepka, Cecilia; Rhodin, Jenny; Lemmens, Raf; Tjerneld, Folke; Gustavsson, Per-Erik c

    2004-01-23

    The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA. PMID:14753711

  8. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems.

    PubMed

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne; Cambon-Bonavita, Marie-Anne

    2014-08-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  9. Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

    PubMed Central

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne

    2014-01-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  10. A comparison of four DNA extraction methods for the detection of Citrus yellow mosaic badna virus from two species of citrus using PCR and dot-blot hybridization

    Microsoft Academic Search

    Basanta K. Borah; A. M. Anthony Johnson; D. V. R. Sai Gopal; Indranil Dasgupta

    2008-01-01

    Nucleic acid preparations extracted using four procedures were assessed to determine the suitability of the procedure for PCR-based and DNA dot-blot-based detection of Citrus yellow mosaic badna virus (CMBV) from two citrus species, acid lime and pummelo. It was found that the success of PCR detection depended upon the procedure of DNA extraction whereas the dot-blot detection was successful with

  11. A rapid semi automated method for DNA extraction from dried-blood spots: Application to the HLA-DR shared epitope analysis in rheumatoid arthritis

    Microsoft Academic Search

    Alexandre Pachot; Véronique Barbalat; Hubert Marotte; Jennifer Diasparra; Aurore Gouraud; Bruno Mougin; Pierre Miossec

    2007-01-01

    Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, “DBS-miniMAG”, that combines filter paper dried blood spots (DBS) with the NucliSens® miniMAG™ semi-automated instrument (bioMérieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in

  12. Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line.

    PubMed

    Almeida, I F; Pinto, A S; Monteiro, C; Monteiro, H; Belo, L; Fernandes, J; Bento, A R; Duarte, T L; Garrido, J; Bahia, M F; Sousa Lobo, J M; Costa, P C

    2015-03-01

    Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoaging. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1?g/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on (1)O2 mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1?g/mL. This effect was found to be related to a direct antioxidant effect (involving (1)O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1?g/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin. PMID:25686820

  13. Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

    Microsoft Academic Search

    Dario De Medici; Luciana Croci; Elisabetta Delibato; Simona Di Pasquale; Emma Filetici; Laura Toti

    2003-01-01

    The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR

  14. Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples

    Microsoft Academic Search

    A. Ramesh; B. P. Padmapriya; A. Chrashekar; M. C. Varadaraj

    2002-01-01

    The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were

  15. 32 P-POSTLABELING AND HPLC SEPARATION OF DNA ADDUCTS FORMED BYDIESEL EXHAUST EXTRACTS IN VITRO AND IN MOUSE SKIN AND LUNG AFTERTOPICAL TREATMENT

    EPA Science Inventory

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form PAH- and nitrated-PAH-DNA adducts in rodent skin and lung. he aim of this study was to characterize by "P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro...

  16. Non-enzymatic hydrolyses of aryl esters of thymidine 3'-monophosphate as probes for the rate-determining step in DNA hydrolysis.

    PubMed

    Takeda, N; Okada, Y; Yashiro, M; Komiyama, M

    1997-01-01

    In order to shed light on the rate-limiting step in DNA hydrolysis, non-enzymatic hydrolyses of aryl esters of thymidine 3'-monophosphate have been kinetically examined. In alkaline solutions, these dinucleotide analogues were hydrolyzed far faster than was thymidylyl(3'-5')thymidine (TpT), indicating that the departure of the leaving group from the phosphorus atom is rate-limiting. The CeIV- and CeIV/PrIII combination-mediated hydrolyses of these analogues were also faster than the corresponding hydrolysis of the dinucleotide. PMID:9586100

  17. Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure

    Microsoft Academic Search

    Tiziana Persico; Valeria Savasi; Enrico Ferrazzi; Monica Oneta; A. E. Semprini; Giuseppe Simoni

    BACKGROUND: To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA\\/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. METHODS: We tested HIV-1 RNA and DNA by nested PCR in semen

  18. An integrated disposable device for DNA extraction and helicase dependent amplification

    E-print Network

    for world health applications, since the need for instrumentation to control flow rate and temperature the polymerase chain reaction (PCR) is the predominant technique of nucleic acid amplification for DNA (or RNA numerous cycles of DNA denaturation, primer annealing, and primer extension. However, instead of heat

  19. Original article Ribosomal DNA and chloroplast DNA

    E-print Network

    Boyer, Edmond

    extraction and analysis The method of total DNA extraction and analysis of cpDNA variation has been described on branches collected in winter and forced in the greenhouse later in the spring. DNA was also extracted fromOriginal article Ribosomal DNA and chloroplast DNA polymorphisms in a mixed stand of Quercus robur

  20. One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold nanoparticles from Memecylon umbellatum

    PubMed Central

    Arunachalam, Kantha D; Annamalai, Sathesh Kumar; Hari, Shanmugasundaram

    2013-01-01

    In this experiment, green-synthesized silver and gold nanoparticles were produced rapidly by treating silver and gold ions with an extract of Memecylon umbellatum leaf. The reaction process was simple and easy to handle, and was monitored using ultraviolet-visible spectroscopy. The effect of the phytochemicals present in M. umbellatum, including saponins, phenolic compounds, phytosterols, and quinones, on formation of stable silver and gold nanoparticles was investigated by Fourier-transform infrared spectroscopy. The morphology and crystalline phase of the nanoparticles were determined by transmission electron microscopy and energy-dispersive x-ray spectroscopy. The results indicate that the saponins, phytosterols, and phenolic compounds present in the plant extract play a major role in formation of silver and gold nanoparticles in their respective ions in solution. The characteristics of the nanoparticles formed suggest application of silver and gold nanoparticles as chemical sensors in the future. Given the simple and eco-friendly approach for synthesis, these nanoparticles could easily be commercialized for large-scale production. PMID:23569372