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1

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure.  

PubMed

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples. PMID:22653603

Zhang, S S; Chen, D; Lu, Q

2012-05-21

2

DNA Extraction  

NSDL National Science Digital Library

Teachers' Domain presents this interactive, adapted from the University of Nebraska's Plant and Soil Science eLibrary, with reading material and animations to help students learn the basics of DNA extraction. The lesson is divided into and introduction and the four processes involved: cell lysis, dismantling the cell membrane, removing unwanted cell parts, and precipitating the DNA. On the site, visitors will also find a supplemental background essay, discussion questions, and standards alignment from Teachers' Domain.

2010-10-07

3

Extracting DNA  

NSDL National Science Digital Library

This lesson for students in grades 9-12 introduces DNA, genes, chromosomes, the chemicals that make up DNA. After the basic information, students will do an experiment in which they will separate out DNA from peas. Knowing that DNA can be separated will give them a base of understanding for future lessons in biology, evolution, biotechnology, and health technology.

Science Netlinks;

2002-03-28

4

FAST, EASY AND EFFICIENT DNA EXTRACTION AND ONE-STEP POLYMERASE CHAIN REACTION FOR THE DETECTION OF XYLELLA FASTIDIOSA IN POTENTIAL INSECT VECTORS  

Technology Transfer Automated Retrieval System (TEKTRAN)

A quick, simple and efficient procedure for detecting Xylella fastidiosa in potential insect vectors is described. The procedure employs a commercially available DNeasy tissue kit for the extraction of high-quality DNA from the insect, followed by one-step polymerase chain reaction amplification usi...

5

Yeast DNA Extraction  

NSDL National Science Digital Library

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.

Hays, Lana

2009-01-01

6

Stepping stones in DNA sequencing  

PubMed Central

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics.

Stranneheim, Henrik; Lundeberg, Joakim

2012-01-01

7

Phytoplasma plasmid DNA extraction.  

PubMed

Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA. PMID:22987431

Andersen, Mark T; Liefting, Lia W

2013-01-01

8

DNA Extraction from Activated Sludges  

Microsoft Academic Search

.   To optimize the cell lysis step for DNA extraction from activated sludge samples, two floc dispersion methods (sonication\\u000a versus stirring with a cation exchange resin), and three cell lysis treatments (lysozyme?+?SDS, sonication in a water bath,\\u000a and thermal shock) were tested. For dispersion, stirring with cation exchange resin was more efficient than sonication. The\\u000a cell lysis procedures were applied

Muriel Bourrain; Wafa Achouak; Vincent Urbain; Thierry Heulin

1999-01-01

9

Fruitful DNA Extraction  

NSDL National Science Digital Library

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!

Kalamuck, Karen; Exploratorium

2000-01-01

10

Automated DNA extraction for large numbers of plant samples.  

PubMed

The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions. PMID:22987412

Mehle, Nataša; Nikoli?, Petra; Rupar, Matevž; Boben, Jana; Ravnikar, Maja; Dermastia, Marina

2013-01-01

11

Event extraction for DNA methylation  

PubMed Central

Background We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. Results We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Conclusions Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

2011-01-01

12

DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP  

PubMed Central

Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells.

Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

2009-01-01

13

Extracting the Max From a DNA Extraction  

NSDL National Science Digital Library

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.

Mulvihill, Charlotte; Bell, Don; Marek, Edmund

2009-01-01

14

Extraction of DNA from soil  

Microsoft Academic Search

There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic

Patrick Robe; Renaud Nalin; Carmela Capellano; Timothy M. Vogel; Pascal Simonet

2003-01-01

15

Extracting DNA from a Banana  

NSDL National Science Digital Library

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.

Gallo, Mark; Ventresca, Shannon; Cordts, Marcia

2012-01-01

16

DNA Extraction Techniques for Use in Education  

ERIC Educational Resources Information Center

|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…

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

2010-01-01

17

A two-step electrodialysis method for DNA purification from polluted metallic environmental samples.  

PubMed

Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples. PMID:18601228

Rodríguez-Mejía, José Luis; Martínez-Anaya, Claudia; Folch-Mallol, Jorge Luis; Dantán-González, Edgar

2008-08-01

18

An efficient protocol for genomic DNA extraction from Citrus species  

Microsoft Academic Search

We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide\\u000a levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using\\u000a water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After\\u000a being washed with

Yun-Jiang Cheng; Wen-Wu Guo; Hua-Lin Yi; Xiao-Min Pang; Xiuxin Deng

2003-01-01

19

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security  

Microsoft Academic Search

Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom

Peter Zimmermann; Kai Vollack; Barbara Haak; Michelle Bretthauer; Andrea Jelinski; Marga Kugler; Jessica Loidl; Werner Pflug

2009-01-01

20

Comparison of Five Simple Methods for DNA Extraction from Echinococcus granulosus Protoscoleces for PCR Amplification of Ribosomal DNA  

Microsoft Academic Search

Background: Cystic hydatid disease is an important zoonosis, affecting humans and animals and is a significant public health and economic problem throughout the world and Iran. Since extraction of DNA from the parasite is a primary and crucial step which has a principal effect on PCR results, in the current study five simple methods for DNA extraction from protoscoleces of

EB Kia; M Fasihi Harandi; F Zahabiun; H Mirhendi

21

Methods for DNA extraction from Candida albicans.  

PubMed

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases. PMID:2823631

Glee, P M; Russell, P J; Welsch, J A; Pratt, J C; Cutler, J E

1987-07-01

22

Step-wise supercritical extraction of carbonaceous residua  

DOEpatents

A method of fractionating a mixture containing high boiling carbonaceous material and normally solid mineral matter which includes processing with a plurality of different supercritical solvents is described. 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.

Warzinski, R.P.

1986-05-15

23

Single-step Charge Transport through DNA over Long Distances  

PubMed Central

Quantum yields for charge transport across adenine tracts of increasing length have been measured by monitoring hole transport in synthetic oligonucleotides between photoexcited 2-aminopurine, a fluorescent analogue of adenine, and N2-cyclopropyl guanine. Using fluorescence quenching, a measure of hole injection, and hole trapping by the cyclopropyl guanine derivative, we separate the individual contributions of single- and multi-step channels to DNA charge transport, and find that with 7 or 8 intervening adenines the charge transport is a coherent, single-step process. Moreover, a transition occurs from multi-step to single-step charge transport with increasing donor/acceptor separation, opposite to that generally observed in molecular wires. These results establish that coherent transport through DNA occurs preferentially across 10 base pairs, favored by delocalization over a full turn of the helix.

Genereux, Joseph C.; Wuerth, Stephanie M.; Barton, Jacqueline K.

2011-01-01

24

Interlaboratory evaluation of the ISO standard 11063 “Soil quality — Method to directly extract DNA from soil samples”  

Microsoft Academic Search

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

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

2011-01-01

25

DNA extraction from keratin and chitin.  

PubMed

DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purification using either silica-column or organic methods. PMID:22237520

Campos, Paula F; Gilbert, Thomas M P

2012-01-01

26

Fern spore extracts can damage DNA  

PubMed Central

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. © 2000 Cancer Research Campaign

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

2000-01-01

27

One-step isothermal assembly of DNA fragments.  

PubMed

The One-Step Isothermal DNA Assembly method allows for the efficient assembly of DNA constructs using fragments up to several hundred kilobases in as little as 15 min. Applications of this method range from the addition of promoters to expression constructs to the assembly of bacterial genome fragments. The production of circularized DNA using this method also enables the direct transformation of target organisms, bypassing intermediate transformations for plasmid propagation in those species where expression could lead to toxicity and cell death. Variations of the method allow for specific cloning tasks to be performed, as well as the use of microarray slides as a source of DNA. The level of precision and simplicity of this method makes it a valuable tool for most cloning efforts and all levels of proficiency in molecular biology. PMID:23996438

Rodrigues, Rui T L; Bayer, Travis S

2013-01-01

28

Microscope Titration and Extraction of DNA from Liver.  

ERIC Educational Resources Information Center

|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)|

Mayo, Lois T.; And Others

1993-01-01

29

Microscope Titration and Extraction of DNA from Liver.  

ERIC Educational Resources Information Center

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)

Mayo, Lois T.; And Others

1993-01-01

30

Human DNA extraction methods: patents and applications.  

PubMed

Since the pioneer experiments conducted by Friedrich Miescher in 1861, extraordinary advances have been achieved in the field of DNA handling. Today nucleic acids can be extracted from any type of biological material such as tissues, cells and viruses. Moreover, increasing knowledge of human genome is paving the way to an effective employment of pharmacogenomics and genetic-based predictive tests in medicine. In this context, the recovery of DNA from different sources of biological samples (e.g. archived formalin-fixed autopsy tissues, dried blood spots, frozen serum or plasma, long-term stored whole blood) is also an emerging field in genetic epidemiology studies. Thus, given the crucial role played by DNA in bio-medical research and in its related applications, here we review the main relevant issued patents and recently published advances in the field of DNA extraction and purification from human specimens. PMID:21303346

Carpi, Francesco M; Di Pietro, Fabio; Vincenzetti, Silvia; Mignini, Fiorenzo; Napolioni, Valerio

2011-04-01

31

Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome  

Microsoft Academic Search

BackgroundDNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.Methodology\\/Principal FindingsIn this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a

Sanqing Yuan; Dora B. Cohen; Jacques Ravel; Zaid Abdo; Larry J. Forney

2012-01-01

32

Loading and activation of DNA replicative helicases: the key step of initiation of DNA replication  

PubMed Central

Evolution has led to diversification of all living organisms from a common ancestor. Consequently, all living organisms use a common method to duplicate their genetic information and thus pass on their inherited traits to their offspring. To duplicate chromosomal DNA, double-stranded DNA must first be unwound by helicase, which is loaded to replication origins and activated during the DNA replication initiation step. In this review, we discuss the common features of, and differences in, replicative helicases between prokaryotes and eukaryotes.

Li, Yan; Araki, Hiroyuki

2013-01-01

33

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...

34

DNA replication in cell-free extracts from Drosophila melanogaster.  

PubMed Central

We have developed an efficient in vitro replication system from 0-2 h Drosophila melanogaster embryos. Demembranated Xenopus sperm DNA when incubated in such an extract first becomes enclosed in a nucleus-like structure with a nuclear envelope and a karyoskeleton. It then undergoes one round of semiconservative replication--this replication appears completely dependent on nuclear formation. Up to 30% of input DNA is nucleated in one reaction. Efficient nuclear formation and replication are dependent on a cold treatment step, prior to disruption of the embryos. They also depend on the age of the embryos used. Extracts from older embryos (0-5 h) are capable of nuclear formation, although at a much reduced efficiency, and repair synthesis, but seem to have lost the ability to initiate DNA replication. In addition to replicating sperm DNA this system appears capable of carrying out semi-conservative replication on some plasmids. However, it cannot use these to trigger nuclear formation; replication is only seen if the plasmids are coincubated with sperm DNA. The in vitro formed nuclei have not been observed to trigger nuclear envelope breakdown and entry into mitosis. However, they can re-replicate the DNA if the nuclei are permeabilized. This system should be a useful complement to the previously isolated Xenopus in vitro replication system. In addition the amenability of Drosophila to genetic study should open up new approaches not previously possible with Xenopus. Images

Crevel, G; Cotterill, S

1991-01-01

35

Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles.  

PubMed

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and cost-effective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications. PMID:19903448

Shan, Zhi; Wu, Qi; Wang, Xianxiang; Zhou, Zhongwu; Oakes, Ken D; Zhang, Xu; Huang, Qianming; Yang, Wanshen

2009-11-10

36

The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twentysomething years on  

Microsoft Academic Search

Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the

Piotr Chomczynski; Nicoletta Sacchi

2006-01-01

37

Robust CTAB-activated charcoal protocol for plant DNA extraction  

Microsoft Academic Search

DNA extracted from plants rich in polyphenols and\\/or polysaccharides is often problematic when subjected to polymerase chain reaction, especially when ma ture tissues are used for DNA extraction. In order to overcome the problems associated with poor-quality DNA extracted from such plant samples, a protocol has been developed, availing on a high salt concentration and on the combination of polyvinylpyrrolidone

Dea BARI?EVI?; Branka JAVORNIK

2006-01-01

38

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

PubMed

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

Verma, Digvijay; Satyanarayana, T

2011-04-26

39

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

PubMed Central

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.

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

2011-01-01

40

A rapid and efficient assay for extracting DNA from fungi  

USGS Publications Warehouse

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.

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

2002-01-01

41

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

Microsoft Academic Search

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

Digvijay Verma; T. Satyanarayana

42

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

Microsoft Academic Search

Polymerase chain reaction (PCR) inhibitors are often co-extracted with ancient DNA (aDNA) and when present make the analysis of aDNA difficult, if not impossible. In this study we review previous research on PCR inhibitors and techniques that address their co-extraction with DNA from sub-optimal samples. Additionally, we introduce a simple extraction technique, “repeat silica extraction,” that effectively removed PCR inhibitors

Brian M. Kemp; Cara Monroe; David Glenn Smith

2006-01-01

43

Human DNA extraction from empty puparia.  

PubMed

Empty puparia, as well as larvae at different developmental stages, are potentially useful in the identification of a victim where a corpse has been removed from the scene of a forensic investigation. To evaluate the relevance and the reliability of this substrate to be used as forensic evidence, the authors report for the first time the extraction and typing of human DNA from empty puparia using STR analysis, in two actual cases where the bodies of the victims were still present thereby enabling validation of the typing. PMID:23821788

Marchetti, Daniela; Arena, Elisa; Boschi, Ilaria; Vanin, Stefano

2013-04-23

44

Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extraction.  

PubMed

The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles-assisted extraction and compared with reverse micellar extraction. The reverse micelles-assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25-fold, respectively. Whereas reverse micelles-assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39-fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles also evidenced that higher purification was obtained in reverse micelles-assisted extraction as compared of reverse micellar extracted lactoperoxidase. PMID:20187078

Nandini, K E; Rastogi, Navin K

45

A Simple Automated Instrument for DNA Extraction in Forensic Casework  

Microsoft Academic Search

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

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

2005-01-01

46

One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.  

PubMed

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 nonspecific 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 30min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly. PMID:23911528

Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

2013-07-31

47

Optimization of DNA extraction from a scleractinian coral for the detection of thymine dimers by immunoassay.  

PubMed

Ultraviolet (UV)-B is known to cause DNA damage, principally by the formation of thymine dimers, but little research has been conducted in coral reef environments where UV doses are high. The majority of tropical reef-dwelling corals form a mutualistic symbiosis with the dinoflagellate Symbiodinium but few studies have been conducted on in situ DNA damage in corals and none have investigated the symbiotic components separately. The aim of this research was to quantify DNA damage in both the coral host and the dinoflagellate symbiont. The first step in this investigation was to optimize the extraction of DNA from the host, Porites astreoides, as well as the symbiont. The optimization was divided into a series of steps: the preservation of the samples, separation of the coral tissue from the skeleton, separation of the host tissue from the algal cells to prevent cross contamination as well as the extraction and purification of genomic DNA from the algae that are located intracellularly within the invertebrate animal tissue. The best preservation method was freezing at low temperatures without ethanol. After scraping with a razor blade, the coral tissue can be divided into host and algal components and the DNA extracted using modifications of published techniques yielding DNA suitable for the quantification of thymine dimer formation using antibodies. Preliminary data suggest that in P. astreoides collected from 1 m depth, thymine dimers form approximately 2.8 times more frequently in the host DNA than in the DNA of its symbionts. PMID:17645654

Banaszak, Anastazia T

48

Extracting Step Stiffness from Correlation Functions Consistent with the GWD  

NASA Astrophysics Data System (ADS)

The statistics of a pair of neighboring steps can be derived approximately from a two-particle Hamiltonian in which the steps are mapped onto spinless fermions. Under typical circumstances, this mapping results in the two-particle Calogero model, for which the complete set of eigenvalues and eigenfunctions can be obtained. The terrace-width distribution (TWD) resulting from this approximation is the ``generalized Wigner distribution'' (GWD), which has been shown to be in excellent agreement with simulations of the terrace-step-kink (TSK) model and to be a useful approximation for experimental TWDs. Here we discuss the use of the complete eigensolution to obtain information about the correlations of step fluctuations along the direction parallel to the step. Of particular interest is the autocorrelation function for the midpoint between adjacent steps. An improved estimate for step stiffness can be derived from this correlation function. The results are compared with preliminary Monte Carlo simulations of the TSK model and with previous Gruber-Mullins predictions.

Benson, Amber; Richards, Howard

2003-03-01

49

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.).  

PubMed

Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods--namely three DNA isolation protocols and three commercial DNA isolation kits--were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs. PMID:22911604

Akkurt, M

2012-08-13

50

One-step cloning and chromosomal integration of DNA.  

PubMed

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids. PMID:24050148

St-Pierre, François; Cui, Lun; Priest, David G; Endy, Drew; Dodd, Ian B; Shearwin, Keith E

2013-05-20

51

A simple Chelex protocol for DNA extraction from Anopheles spp.  

PubMed

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 ?l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 ?l was retained while the other 10 ?l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9). Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality, a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain. PMID:23328684

Musapa, Mulenga; Kumwenda, Taida; Mkulama, Mtawa; Chishimba, Sandra; Norris, Douglas E; Thuma, Philip E; Mharakurwa, Sungano

2013-01-09

52

DNA Extraction Procedures Meaningfully Influence qPCR-Based mtDNA Copy Number Determination  

PubMed Central

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA: nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA: nDNA ratios in genomic DNA samples prepared using organic solvent (phenol-chloroform-isoamylalcohol) extraction and two different silica-based column methods, and found mtDNA: nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA: nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.

Guo, Wen; Jiang, Lan; Bhasin, Shalender; Khan, Shaharyar M.; Swerdlow, Russell H.

2009-01-01

53

A comparison of DNA extraction methods using Petunia hybrida tissues.  

PubMed

Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields. PMID:23997658

Tamari, Farshad; Hinkley, Craig S; Ramprashad, Naderia

2013-09-01

54

Integrated lysis procedures reduce extraction biases of microbial DNA from mangrove sediment  

Microsoft Academic Search

Sufficient lysis of soil or sediment microbes is a critical step for analyzing microbial community structures and for preparing metagenomic DNA libraries. The present study compared lysis methods for recovering archaeal, bacterial, actinomycete, and fungal DNAs from a mangrove sediment sample. PCR results showed that individual procedures using SDS, lysozyme, sonication, freeze–thaw, microwave, and vigorous shaking could extract archaeal or

Yun-Xia Jiang; Ji-Guo Wu; Ke-Qiang Yu; Chun-Xiang Ai; Fei Zou; Hong-Wei Zhou

2011-01-01

55

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 ...

56

Extraction of High Molecular Weight DNA from Microbial Mats  

PubMed Central

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 ?g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

Bey, Benjamin S.; Fichot, Erin B.; Norman, R. Sean

2011-01-01

57

Extremely Rapid Extraction of DNA from Bacteria and Yeasts  

Microsoft Academic Search

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic

Hai-Rong Cheng; Ning Jiang

2006-01-01

58

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

Microsoft Academic Search

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

David N. Fredricks; Caitlin Smith; Amalia Meier

2005-01-01

59

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

PubMed Central

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.

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

60

Giardia intestinalis: DNA extraction approaches to improve PCR results.  

PubMed

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol. PMID:21315715

Babaei, Zahra; Oormazdi, Hormozd; Rezaie, Sasan; Rezaeian, Mostafa; Razmjou, Elham

2011-02-17

61

A plant proteinase, extracted from Bromelia fastuosa, as an alternative to proteinase K for DNA extraction  

Microsoft Academic Search

We report the use of brofasin, a proteolytic enzyme extracted from the fruits of Bromelia fastuosa, as a protective agent in DNA extraction protocols. Brofasin at concentrations varying between 236 units\\/µg to 2,372 units\\/µg was used to extract DNA of human leukocytes and Drosophila melanogaster tissues. At brofasin concentration between 1,186 and 2,372 units\\/µg the resulting DNA concentrations are similar

M. T. Ruiz; C. M. A. Carareto; G. O. Bonilla-Rodriguez; Gustavo O. Bonilla Rodriguez

62

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

Microsoft Academic Search

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

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

2009-01-01

63

A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes.  

PubMed

A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA. PMID:16691008

Tapia-Tussell, Raul; Lappe, Patricia; Ulloa, Miguel; Quijano-Ramayo, Andrés; Cáceres-Farfán, Mirbella; Larqué-Saavedra, Alfonso; Perez-Brito, Daisy

2006-05-01

64

Safer DNA extraction from plant tissues using sucrose buffer and glass fiber filter.  

PubMed

For some plant species, DNA extraction and downstream experiments are inhibited by various chemicals such as polysaccharides and polyphenols. This short communication proposed an organic-solvent free (except for ethanol) extraction method. This method consists of an initial washing step with STE buffer (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA), followed by DNA extraction using a piece of glass fiber filter. The advantages of this method are its safety and low cost. The purity of the DNA solution obtained using this method is not necessarily as high as that obtained using the STE/CTAB method, but it is sufficient for PCR experiments. These points were demonstrated empirically with two species, Japanese speedwell and common dandelion, for which DNA has proven difficult to amplify via PCR in past studies. PMID:22695723

Takakura, Koh-Ichi; Nishio, Takayuki

2012-06-14

65

Comparison of DNA and RNA extraction methods for mummified tissues  

Microsoft Academic Search

Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were

Nami Konomi; Eve Lebwohl; David Zhang

2002-01-01

66

Terrace-width distributions on vicinal surfaces: generalized Wigner surmise and extraction of step step repulsions  

NASA Astrophysics Data System (ADS)

From quantitative measurement of the equilibrium terrace-width (?) distribution (TWD) of vicinal surfaces, one can assess the strength A of elastic step-step repulsions A/? 2. Generally the TWD depends only on Ã=A×( step stiffness )/(k BT) 2. From ideas of fluctuation phenomena, TWDs should be describable by the "generalized Wigner distribution" (GWD), essentially a power-law in ?/ times a "Gaussian decay" in ?/. The power-law exponent is related simply to Ã. Alternatively, the GWD gives the exact solution for a mean-field approximation. The GWD provides at least as good a description of TWDs as the standard fit to a Gaussian (centered at ). It works well for weak elastic repulsion strengths à (where Gaussians fail), as illustrated explicitly for vicinal Pt(1 1 0). Application to vicinal copper surfaces confirms the viability of the GWD analysis. The GWD can be treated as a two-parameter fit by scaling ? using an adjustable characteristic width. With Monte Carlo and transfer-matrix calculations, we show that for physical values of Ã, the GWD provides a better overall estimate than the Gaussian models. We quantify how a GWD approaches a Gaussian for large à and present a convenient, accurate expression relating the variance of the TWD to Ã. We describe how discreteness of terrace widths impacts the standard continuum analysis.

Einstein, T. L.; Richards, Howard L.; Cohen, Saul D.; Pierre-Louis, O.; Giesen, M.

2001-05-01

67

Study of the DNA damage checkpoint using Xenopus egg extracts.  

PubMed

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, ?-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure. PMID:23149695

Willis, Jeremy; DeStephanis, Darla; Patel, Yogin; Gowda, Vrushab; Yan, Shan

2012-11-05

68

Automated Extraction of DNA from Forensic Sample Types Using the PrepFiler Automated Forensic DNA Extraction Kit  

Microsoft Academic Search

The HID EVOlution—Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SPI

Maxim Brevnov; Janna Mundt; Jacki Benfield; Lynda Treat-Clemons; Geert Kalusche; Jason Meredith; Gregory Porter; Manohar R. Furtado; Jaiprakash G. Shewale

2009-01-01

69

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

PubMed

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

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

2011-06-22

70

Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories.  

PubMed

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities, their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic DNA Collection in order to address specific health priorities through molecular techniques. Our approach employs a low-cost laundry detergent based DNA extraction technique that maximizes DNA yield and quality. We have optimised four different modalities (maxiprep, midiprep, miniprep and microprep) for two different sources (leukocyte concentrates and whole blood). Our optimised protocol produces 4.5 mg of DNA from 15 ml of blood-bank discarded leukocyte concentrates with spectrophotometric quality, genomic integrity and PCR suitability that rivals that of phenol-chloroform extracted samples. We present evidence of many PCR applications that we have carried out on samples extracted with this technique including Killer-cell Immunoglobulin-like Receptor genotyping, Short Tandem Repeat profiling as well as nucleic acid screening for hepatitis B and human immunodeficiency type-1 viruses. This paper highlights many of the advantages that this DNA extraction technique provides over existing methodologies, whether it is used to establish large genomic DNA collections (as was our main intention) or as a routine DNA extraction method for PCR applications. PMID:19609718

Garcia-Sepulveda, Christian Alberto; Carrillo-Acuña, Enrique; Guerra-Palomares, Sandra Elizabeth; Barriga-Moreno, Montserrat

2009-07-17

71

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

Microsoft Academic Search

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

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

2012-01-01

72

Optimization of DNA Extraction from Deep-sea Basalt  

NASA Astrophysics Data System (ADS)

Studies on the microorganisms that inhabit deep-sea basalt can provide information on this dark ecosystem, which will contribution to our understanding of mass transformation and energy flow in the deep ocean. However, molecular methods for use with metal- and clay-rich rock materials such as basalt have not been suitably developed at present, yet are critically required in order to be able to fully evaluate the basalt biotope. For example, inefficient DNA extraction might lead to loss of information about important components of this community, and misinterpretation about the total community diversity and function. In order to investigate the effects of sample pretreated method, particle size, different DNA extraction methods and cell density on extracted DNA yields, two basalt samples were collected from the East Pacific Rise 9° N during research cruise AT11- 20 in Nov 2004. Basalt samples were crushed to different particle size, washed with ddH2O and 100% ethanol respectively, and autoclaved. Marinobacter aquaeolei cultures with different cell densities were inoculated into differently treated basalt samples. Pure culture and basalt samples without inoculation were used as positive and negative control to evaluate the extracting efficiency. FastDNA spin for soil kit, GeneClean for ancient DNA kit and UltraCleanTM soil DNA Kit are used for DNA extraction. Results showed that DNA yields increased with culture density. FastDNA spin for soil kit gave the highest DNA yields, which is almost 10 times more than that of UltraCleanTM soil DNA Kit. Ethanol washing and ddH2O washing did not make big difference to DNA yields. Mineral composition and surface areas might also affect DNA yields.

Wang, H.; Edwards, K. J.

2007-12-01

73

DNA extraction and amplification from Giemsa-stained blood smears.  

PubMed

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

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

1995-01-01

74

Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome  

PubMed Central

Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

Yuan, Sanqing; Cohen, Dora B.; Ravel, Jacques; Abdo, Zaid; Forney, Larry J.

2012-01-01

75

Incorporation of CC Steps into Z-DNA: Interplay Between B/Z Junction and Z-DNA Helical Formation  

PubMed Central

The left-handed DNA structure, Z-DNA, is believed to play important roles in gene expression and regulation. Z-DNA forms sequence-specifically with a preference for sequences rich in pyrimidine/purine dinucleotide steps. In vivo, Z-DNA is generated in the presence of negative supercoiling or upon binding proteins that absorb the high energetic cost of the B-to-Z transition including the creation of distorted junctions between B-DNA and Z-DNA. To date, the sequence-preferences for the B-to-Z transition have primarily been studied in the context of sequence repeats lacking B/Z junctions. Here, we develop a method to characterize sequence-specific preferences for Z-DNA formation and B/Z junction localization within heterogeneous DNA duplexes that is based on combining 2-aminopurine fluorescence measurements with a new quantitative application of CD spectroscopy for determining the fraction of B- versus Z-DNA. Using this approach, we show that at least three consecutive CC dinucleotide steps, traditionally thought to disfavor Z-DNA, can be incorporated within heterogeneous Z-DNA containing B/Z junctions upon binding to the Z? domain of the RNA adenosine deaminase (ADAR1) protein. Our results indicate that the incorporation of CC steps into Z-DNA is driven by favorable sequence-specific Z/Z and B/Z stacking interactions as well as by sequence-specific energetics that localize the distorted B/Z junction at flexible sites. Together, our results expose higher order complexities in the Z-DNA code within heterogeneous sequences and suggest that Z-DNA can in principle propagate into a wider range of genomic sequence elements than previously thought.

Bothe, Jameson R.; Lowenhaupt, Ky; Al-Hashimi, Hashim M.

2013-01-01

76

DNA extraction method from bones using Maxwell® 16.  

PubMed

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

Karija Vlahovi?, Monika; Kubat, Milovan

2012-05-23

77

Comparison of DNA extraction methods in analysis of salivary bacterial communities.  

PubMed

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

Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

2013-07-03

78

Damping of betatron oscillations in the SPS appearing at the two step extraction of CNGS beam  

Microsoft Academic Search

The two batches comprising the CNGS beam in the SPS will be extracted towards the target in two steps separated in time by 50 ms (about 2170 turns). Since the kicker pulse used to extract the first batch is not ideal, the second batch will suffer from some residual kick and will oscillate transversely. These oscillations will prevent the second

E Vogel; Wolfgang Höfle; E Gaxiola

2005-01-01

79

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

Microsoft Academic Search

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.

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

2007-01-01

80

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

PubMed

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 which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene. PMID:16099520

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

2005-08-15

81

An improved extraction method to increase DNA yield from molted ...  

Treesearch

We also compared PCR success across the same five feather types using five ... Although our modified extraction method increased the time required for ... it resulted in significantly higher yields of DNA as compared to the unmodified protocol.

82

An improved extraction method to increase DNA yield from molted ...  

Treesearch

Jul 21, 2013 ... We also compared PCR success across the same five feather types using five ... Although our modified extraction method increased the time required for ... higher yields of DNA as compared to the unmodified protocol.

83

Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis  

Microsoft Academic Search

Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which

Bram Bekaert; Maarten H. D. Larmuseau; Maarten P. M. Vanhove; Anouschka Opdekamp; Ronny Decorte

84

Numerical extraction of Cole-Cole impedance parameters from step response  

NASA Astrophysics Data System (ADS)

In this paper we propose a numerical method to extract the four parameters (Ro, R?, C, and ?) that characterize a single-dispersion Cole-Cole impedance model from its step response, based on modelling the step response using fractional calculus, without requiring direct measurement of the real and imaginary impedance parts. MATLAB simulations using Cole-Cole impedances of fruit tissues with ?<1, PSPICE simulations and experimental results using a Cole-Cole model with ?=1 are given to verify the method. Extracted impedance parameters show less than 0.1% relative error in simulation and less than 10% error in experimental results for the extracted impedance parameters.

Freeborn, Todd J.; Maundy, Brent; Elwakil, Ahmed

85

High efficiency DNA extraction from bone by total demineralization.  

PubMed

In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol. PMID:19083754

Loreille, Odile M; Diegoli, Toni M; Irwin, Jodi A; Coble, Michael D; Parsons, Thomas J

2007-03-12

86

Methods of sperm DNA extraction for genetic and epigenetic studies.  

PubMed

High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR. PMID:22992929

Griffin, Jeanine

2013-01-01

87

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

Microsoft Academic Search

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

Svante Paabo

1989-01-01

88

Extraction of high quality DNA from seized moroccan cannabis resin (hashish).  

PubMed

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances. PMID:24124454

El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaâ; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-10-04

89

Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)  

PubMed Central

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances.

El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaa; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-01-01

90

Toward DNA-based Security Circuitry: First Step - Random Number Generation  

PubMed Central

DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. Our team investigates the implications of DNA-based circuit design in serving security applications. As an initial step we develop a random number generation circuitry. A novel prototype schema employs solid-phase synthesis of oligonucleotides for random construction of DNA sequences. Temporary storage and retrieval is achieved through plasmid vectors.

Bogard, Christy M.; Arazi, Benjamin; Rouchka, Eric C.

2010-01-01

91

Patterning of a nanoporous membrane for multi-sample DNA extraction  

NASA Astrophysics Data System (ADS)

A novel method for patterning a nanoporous aluminum oxide membrane was developed using SU-8 to allow for extraction of DNA from multiple samples simultaneously. To facilitate the patterning process, the spin curve for SU-8 2015 on the membrane was determined. The correlation profile of the SU-8 spin curve was similar in form to curves of SU-8 spun on silicon wafers, but with a uniform decrease in thickness across the curve. Characterization of the patterned SU-8 regarding step height and surface roughness was determined using pin-based surface profilometry. The patterned aluminum oxide membrane displayed acceptable flatness and precise pattern replication. Thickness uniformity was also observed to be approximately ±2.8% for a 29.3 µm thick pattern. SEM imaging was used to examine the exposed membrane surface for any visible changes or damage to the membrane caused by the patterning process, with none observed. DNA extraction was performed on the patterned membrane using multiple wells to show the ability of collecting multiple DNA samples simultaneously. DNA, marked with a fluorescent dye, was collected on the membrane and successfully observed using a fluorescence microscope. The techniques developed in this work have application to lab-on-a-chip type systems that include DNA extraction steps.

Kim, Jungkyu; Voelkerding, Karl V.; Gale, Bruce K.

2006-01-01

92

New DNA Extraction Methods for Casework Evidence  

Microsoft Academic Search

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

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

93

Evidence on DNA slippage step-length distribution  

NASA Astrophysics Data System (ADS)

A simple model based on a master equation is constructed in order to reveal the details of the mutational events modifying simple sequence repeats in the human genome, A database of simple repeats together with their flanking sequences comprising approximately 105 entries from all 24 human chromosomes was constructed. By aligning the pairs of fragments of sequences containing the repeat elements, the matrices that count the number of slippage events were evaluated. The counts were then used as a target to be reproduced by our theoretical model, in which the elongation and shortening of the repeats proceed through a mechanism in which the step lengths exhibit a decaying distribution in the form of an inverse power law rather than through one nucleotide extension or deletion, which was the most frequent supposition in previous studies.

Borštnik, Branko; Pumpernik, Danilo

2005-03-01

94

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.  

PubMed

The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended. PMID:22853974

Eland, Lucy E; Davenport, Russell; Mota, Cesar R

2012-07-24

95

Purification of crime scene DNA extracts using centrifugal filter devices  

PubMed Central

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.

2013-01-01

96

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

PubMed

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

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

2008-11-11

97

Rapid and Reliable Method of Extracting DNA and RNA from Sweetpotato, Ipomoea batatas (L). Lam  

Microsoft Academic Search

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg\\/g leaf tissue). The extracted DNA was completely digested by restriction

Sun-Hyung Kim; Tatsuro Hamada

2005-01-01

98

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles  

PubMed Central

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

Lezin, George; Kosaka, Yasuhiro; Yost, H. Joseph; Kuehn, Michael R.; Brunelli, Luca

2011-01-01

99

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

PubMed

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

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

2012-02-05

100

DNA Extraction Columns Contaminated with Murine Sequences  

Microsoft Academic Search

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

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

2011-01-01

101

A DNA extraction method of small quantities of bone for high-quality genotyping.  

PubMed

DNA genotyping techniques have been used successfully in forensic science for almost three decades and represent the gold standard for individual identification. However, efficient protocols for obtaining DNA from exhumed bones suitable for genotyping are still scarce and most of them require a considerable amount of starting material, are time consuming and are inefficient for reducing inhibitor's effects. We sought to develop an optimised protocol for extracting DNA from bone samples obtained from exhumations. We tested two approaches for preparing bone samples: (a) fine powder and (b) thin slices of bone. The best ratio of bone amount to DNA yields was assessed by a titration experiment using bone powder ranging from 50 to 1000mg. We obtained optimal DNA yields (27pgmg(-1) on average) when 150-200mg of starting material were processed using a one-step demineralisation method. Better-quality profiles (determined by the number of genotyped loci) were obtained when DNA was extracted from bone slices compared to extraction from bone powder. From bone slices 83.9% and from bone powder 46.7% of the samples provided genotypes for 11 or more loci. Since bone preparation procedures were carried out at room temperature, the method developed in the present study might be an attractive alternative to the standard freeze-mill approach, being faster and more cost-efficient. PMID:23948318

Caputo, Mariela; Irisarri, Maximiliano; Alechine, Evguenia; Corach, Daniel

2013-06-28

102

Three reversible and controllable discrete steps of channel gating of a viral DNA packaging motor.  

PubMed

The channel of the viral DNA packaging motor allows dsDNA to enter the protein procapsid shell during maturation and to exit during infection. We recently showed that the bacteriophage phi29 DNA packaging motor exercises a one-way traffic property using a channel as a valve for dsDNA translocation. This raises a question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel inward. We proposed that DNA forward or reverse travel is controlled by conformational changes of the channel. Here we reported our direct observation that the channel indeed exercises conformational changes by single channel recording at a single-molecule level. The changes were induced by high electrical voltage, or by affinity binding to the C-terminal wider end located within the capsid. Novel enough, the conformational change of the purified connector channel exhibited three discrete gating steps, with a size reduction of 32% for each step. We investigated the role of the terminal and internal loop of the channel in gating by different mutants. The step-wise conformational change of the channel was also reversible and controllable, making it an ideal nano-valve for constructing a nanomachine with potential applications in nanobiotechnology and nanomedicine. PMID:21807410

Geng, Jia; Fang, Huaming; Haque, Farzin; Zhang, Le; Guo, Peixuan

2011-07-31

103

Extracting information from cDNA arrays  

NASA Astrophysics Data System (ADS)

High-density DNA arrays allow measurements of gene expression levels (messenger RNA abundance) for thousands of genes simultaneously. We analyze arrays with spotted cDNA used in monitoring of expression profiles. A dilution series of a mouse liver probe is deployed to quantify the reproducibility of expression measurements. Saturation effects limit the accessible signal range at high intensities. Additive noise and outshining from neighboring spots dominate at low intensities. For repeated measurements on the same filter and filter-to-filter comparisons correlation coefficients of 0.98 are found. Next we consider the clustering of gene expression time series from stimulated human fibroblasts which aims at finding co-regulated genes. We analyze how preprocessing, the distance measure, and the clustering algorithm affect the resulting clusters. Finally we discuss algorithms for the identification of transcription factor binding sites from clusters of co-regulated genes.

Herzel, Hanspeter; Beule, Dieter; Kielbasa, Szymon; Korbel, Jan; Sers, Christine; Malik, Arif; Eickhoff, Holger; Lehrach, Hans; Schuchhardt, Johannes

2001-03-01

104

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

PubMed

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

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

2012-09-11

105

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

NASA Astrophysics Data System (ADS)

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.

Ho, Anthony; Comer, Jeffrey; Aksimentiev, Aleksei

2011-03-01

106

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform. PMID:21885362

Amory, Sylvain; Huel, René; Bili?, Ana; Loreille, Odile; Parsons, Thomas J

2011-08-31

107

Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories  

Microsoft Academic Search

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve\\u000a statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities,\\u000a their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic\\u000a DNA Collection in order to address specific health priorities through molecular

Christian Alberto Garcia-Sepulveda; Enrique Carrillo-Acuña; Sandra Elizabeth Guerra-Palomares; Montserrat Barriga-Moreno

2010-01-01

108

DNA Extraction from Museum Specimens of Parasitic Hymenoptera  

PubMed Central

At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ?150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation.

Andersen, Jeremy C.; Mills, Nicholas J.

2012-01-01

109

Rapid and effective DNA extraction method with bead grinding for a large amount of fungal DNA.  

PubMed

To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify fungi with molecular techniques, such as DNA fingerprinting. PMID:20537263

Watanabe, M; Lee, K; Goto, K; Kumagai, S; Sugita-Konishi, Y; Hara-Kudo, Y

2010-06-01

110

Cell lysis and DNA extraction in microfabricated devices  

NASA Astrophysics Data System (ADS)

We are developing a microfabricated device to lyse single cells and extract the DNA. The chip consists of two parts: a diffuse mixer combined with a dielectrophoretic trap. We are working with E. coli which have been made osmoticaly unstable before loading into the chip. The cells are lysed by osmotic shock in the mixer. The lysate is then passed to the dielectrophoretic trap. Attempts to separate the genomic DNA from the lysate fragments by selectively trapping the DNA using dielectrophoresis have been made. We have encountered cell sticking problems and are investingating surface modifications using Polyethylene glycol to solve this problem.

Prinz, Christelle; Tegenfeldt, Jonas; Austin, Robert

2002-03-01

111

Comparative analysis of microbial DNA extraction protocols for groundwater samples.  

PubMed

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit. PMID:21683680

Purswani, Jessica; Martín-Platero, Antonio Manuel; Reboleiro-Rivas, Patricia; González-López, Jesús; Pozo, Clementina

2011-05-27

112

Toward an Integrated Microdevice for DNA Extraction and PCR Amplification in the Submicroliter Regime for Forensic DNA Analysis  

Microsoft Academic Search

The purification\\/extraction of DNA from evidentiary material and the amplification of target STR sequences in the purified DNA represent fundamental processes in forensic typing. Integrating these processes on a single microdevice is challenging as a result of the very distinct nature of these two processes. DNA purification, as accomplished by DNA solid phase extraction (SPE), is a chromatographic process carried

Michael G. Roper; Lindsay A. Legendre; Joan M. Bienvenue; Jerome P. Ferrance; James P. Landers

113

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.  

PubMed

The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. PMID:23121735

Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

2012-11-03

114

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

PubMed Central

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

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

1994-01-01

115

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy  

PubMed Central

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

Kim, Jiho; Doose, Soren; Neuweiler, Hannes; Sauer, Markus

2006-01-01

116

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy.  

PubMed

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides. PMID:16687657

Kim, Jiho; Doose, Sören; Neuweiler, Hannes; Sauer, Markus

2006-05-10

117

The role of CDK in the initiation step of DNA replication in eukaryotes  

PubMed Central

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.

Tanaka, Seiji; Tak, Yon-Soo; Araki, Hiroyuki

2007-01-01

118

A DNA extraction and purification method for several yeast genera  

Microsoft Academic Search

Biodiversity and other branches of biological research require rapid and inex- pensive methods to extract and purify DNA, able to process several samples simultaneous- ly, thus allowing an extensive approach to the molecular study of diversity in nature. One problem is the necessity of choosing between rapid methods able to produce relatively crude products or time-consuming procedure yielding nucleic acids

G. CARDINALI; A. BOLANO; A. MARTINI

2001-01-01

119

Direct Extraction and Amplification of DNA from Soil.  

ERIC Educational Resources Information Center

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)

Trevors, Jack T.; Leung, K.

1998-01-01

120

CSP-DNA EXTRACTION & STAGING LABORATORY Prices 2012 & 2013  

Cancer.gov

08/23/13 Frederick National Laboratory for Cancer Research CSP-DNA EXTRACTION & STAGING LABORATORY Services without a price for a given year: may not be available, the price is pending or it hasn't been selected to be displayed on the web. Service

121

Using Concrete & Representational Experiences to Understand the Structure of DNA: A Four-Step Instructional Framework  

ERIC Educational Resources Information Center

|A description of learning experience that uses a four-step instrumentational framework involving concrete and representational experiences to promote conceptual understanding of abstract biological concepts by a series of closely-related activities is presented. The students are introduced to the structure and implications of DNA using four…

Harrell, Pamela Esprivalo; Richards, Debbie; Collins, James; Taylor, Sarah

2005-01-01

122

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX  

Microsoft Academic Search

BACKGROUND: As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here

Andreas Nitsche; Andreas Kurth; Anna Dunkhorst; Oliver Pänke; Hendrik Sielaff; Wolfgang Junge; Doreen Muth; Frieder Scheller; Walter Stöcklein; Claudia Dahmen; Georg Pauli; Andreas Kage

2007-01-01

123

Developmental validation of the PrepFiler Forensic DNA Extraction Kit for extraction of genomic DNA from biological samples.  

PubMed

The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts. PMID:19302383

Brevnov, Maxim G; Pawar, Hemant S; Mundt, Janna; Calandro, Lisa M; Furtado, Manohar R; Shewale, Jaiprakash G

2009-03-16

124

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

Microsoft Academic Search

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

Mohamad Ghazali; Jalan Sultan

125

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

NASA Astrophysics Data System (ADS)

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.

Morales, Mercedes C.

126

Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.  

PubMed

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs. PMID:20849891

Nylund, Lotta; Heilig, Hans G H J; Salminen, Seppo; de Vos, Willem M; Satokari, Reetta

2010-09-16

127

Evaluation of DNA extraction methods of rumen microbial populations.  

PubMed

The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay. PMID:23054703

Villegas-Rivera, Gabriela; Vargas-Cabrera, Yevani; González-Silva, Napoleón; Aguilera-García, Florentino; Gutiérrez-Vázquez, Ernestina; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Baizabal-Aguirre, Víctor Manuel; Valdez-Alarcón, Juan José

2012-10-05

128

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples  

PubMed Central

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Smith, K.; Diggle, M. A.; Clarke, S. C.

2003-01-01

129

One-step purification of metallothionein extracted from two different sources  

Microsoft Academic Search

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and

Rubens T. Honda; Roziete Mendes Araújo; Bruno Brasil Horta; Adalberto L. Val; Marilene Demasi

2005-01-01

130

Effective one/two step purification of various materials by solid-phase extraction.  

PubMed

Simple one/two step purification procedures based on the solid-phase extraction technique were effectively exploited to clean up radiolabelled drugs represented by dihydrochloride of [6-3H]-stobadine and hydrochloride of [4-3H]-pentacaine, derivatization agents such as 4-nitrobenzoyl chloride or 3,5-dinitrobenzoyl chloride, as well as the aqueous phosphate or triethylamine acetate buffer solutions. PMID:9413613

Soltés, L; Sébille, B

131

The Mechanism of the Translocation Step in DNA Replication by DNA Polymerase I: A Computer Simulation Analysis  

PubMed Central

Summary High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O-helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1-helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

Golosov, Andrei A.; Warren, Joshua J.; Beese, Lorena S.; Karplus, Martin

2012-01-01

132

The Mechanism of the Translocation Step in DNA Replication by DNA Polymerase I: A Computer Simulation Analysis  

SciTech Connect

High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

Golosov, Andrei A.; Warren, Joshua J.; Beese, Lorena S.; Karplus, Martin (Harvard); (Duke)

2010-11-03

133

Repair of Damaged DNA by Arabidopsis Cell Extract  

PubMed Central

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light– and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue–induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.

Li, Anatoliy; Schuermann, David; Gallego, Francesca; Kovalchuk, Igor; Tinland, Bruno

2002-01-01

134

Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe  

PubMed Central

DNA base excision repair (BER) accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3? end. The major AP endonuclease Apn2p functions predominantly in removing the 3? block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER). Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5? side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3? block. Other enzymes working in 3? end processing are also discussed.

Kanamitsu, Kyoichiro; Ikeda, Shogo

2010-01-01

135

Evaluation of DNA extraction methods from green and roasted coffee beans  

Microsoft Academic Search

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

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

2008-01-01

136

Single-step multicolor fluorescence In situ hybridization using semiconductor quantum dot-DNA conjugates  

Microsoft Academic Search

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based\\u000a fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization\\/detection\\u000a step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of

Laurent A. Bentolila; Shimon Weiss

2006-01-01

137

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

PubMed

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

Pääbo, S

1989-03-01

138

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

Microsoft Academic Search

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

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

2007-01-01

139

Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs ?  

PubMed Central

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.

Dauphin, Leslie A.; Hutchins, Rebecca J.; Bost, Liberty A.; Bowen, Michael D.

2009-01-01

140

Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood  

PubMed Central

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults.

Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Ieda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

2011-01-01

141

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

PubMed Central

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.

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

2002-01-01

142

Dextran sulfate as a contaminant of DNA extracted from concentrated viruses and as an inhibitor of DNA polymerases  

Microsoft Academic Search

Dextran sulfate is commonly used with polyethylene glycol to concentrate viruses before extraction of their DNA. However, dextran sulfate then easily contaminated such DNA and acted as a potent inhibitor of DNA polymerases from Bacillus subtilis (III), phage PBS2, and phage T4. Dextran sulfate only weakly inhibited Micrococcus luteus and Escherichia coli DNA polymerase I preparations.

R. A. Hitzeman; A. M. Hanel; A. R. Price

1978-01-01

143

DNA charge transport as a first step in coordinating the detection of lesions by repair proteins  

PubMed Central

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. We have proposed a model where repair proteins containing redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5?-3? helicase involved in nucleotide excision repair, contains a [4Fe-4S] cluster and exhibits a DNA-bound redox potential that is physiologically relevant. In AFM studies, we observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. We further provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome.

Sontz, Pamela A.; Mui, Timothy P.; Fuss, Jill O.; Tainer, John A.; Barton, Jacqueline K.

2012-01-01

144

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

PubMed Central

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 knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.

Little, Max A.; Steel, Bradley C.; Bai, Fan; Sowa, Yoshiyuki; Bilyard, Thomas; Mueller, David M.; Berry, Richard M.; Jones, Nick S.

2011-01-01

145

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

PubMed

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 method yields purified extracts from a small sample set (? 2-6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs. PMID:21288791

Hudlow, William R; Buoncristiani, Martin R

2011-02-01

146

Asymmetric Processing of Human Immunodeficiency Virus Type 1 cDNA In Vivo: Implications for Functional End Coupling during the Chemical Steps of DNA Transposition  

PubMed Central

Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3? ends of linear transposon or donor DNA are joined to the 5? phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.

Chen, Hongmin; Engelman, Alan

2001-01-01

147

DNA pairing is an important step in the process of targeted nucleotide exchange  

PubMed Central

Modified single-stranded DNA oligonucleotides can direct the repair of genetic mutations in yeast, plant and mammalian cells. The mechanism by which these molecules exert their effect is being elucidated, but the first phase is likely to involve the homologous alignment of the single strand with its complementary sequence in the target gene. In this study, we establish the importance of such DNA pairing in facilitating the gene repair event. Oligonucleotide-directed repair occurs at a low frequency in an Escherichia coli strain (DH10B) lacking the RECA DNA pairing function. Repair activity can be rescued by using purified RecA protein to catalyze the assimilation of oligonucleotide vectors into a plasmid containing a mutant kanamycin resistance gene in vitro. Electroporation of the preformed complex into DH10B cells results in high levels of gene repair activity, evidenced by the appearance of kanamycin-resistant colonies. Gene repair is dependent on the formation of a double-displacement loop (double-D-loop), a recombination intermediate containing two single-stranded oligonucleotides hybridized to opposite strands of the plasmid at the site of the point mutation. The heightened level of stability of the double-D-loop enables it to serve as an active template for the DNA repair events. The data establish DNA pairing and the formation of the double-D-loop as important first steps in the process of gene repair.

Drury, Miya D.; Kmiec, Eric B.

2003-01-01

148

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

PubMed

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 hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification. PMID:15062955

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

2004-05-10

149

[Analysis of alcoholic extracts of Cistanche deserticola by tri-step identification of Fourier transform infrared spectroscopy].  

PubMed

Tri-step identification of Fourier transform infrared spectroscopy (FTIR) integrated with second derivative spectroscopy and two dimensional correlation infrared spectroscopy (2D-IR) were applied to analyze and evaluate the alcoholic extracts and corresponding residues of cistanche deserticola from the surface to what lies behind. It was found that active compounds including phenylethanoid glycosides were enriched effectively after alcoholic extraction and the extract by using 70% of alcohol had the highest concentration compared to the others. The technique of the tri-step identification holistically disclosed the profile of active compounds in cistanche deserticola extracted by a series of concentrations of ethanol and validated the rationality of the traditional alcoholic extraction method. It not only could be used in monitoring the process of the alcoholic extraction and the compositions of the extracts and residues, discriminating micro-differences among them, but also could provide a macroscopic guidance for medicinal and pharmacological studies. PMID:22242486

Zhang, Sheng-Jun; Xu, Chang-Hua; Chen, Jian-Bo; Zhou, Qun; Sun, Su-Qin

2011-11-01

150

DNA extraction and analysis from processed coffee beans.  

PubMed

The authenticity of coffee is an important issue for both producers and consumers. Premium Arabica material is especially prone to being adulterated, and a number of different techniques have been employed to determine the quality of both roasted and instant coffee. Currently, assessment of coffee authenticity relies on chemical methods which can discriminate between coffee species, but not varieties. Several genetic markers are available for assessing coffee origin, but their suitability to testing commercial coffee is limited by the ability to extract DNA from highly processed beans. In this paper, we demonstrate that PCR-grade DNA may be obtained from roasted beans and even instant coffee. This would allow analysis of commercial samples, provided that suitable markers for species/variety identification are found. PMID:16248533

Martellossi, Chiara; Taylor, Emily J; Lee, David; Graziosi, Giorgio; Donini, Paolo

2005-11-01

151

Akonni TruTip(®) and Qiagen(®) methods for extraction of fetal circulating DNA--evaluation by real-time and digital PCR.  

PubMed

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

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

2013-08-06

152

Indirect Readout of DNA Sequence at the Primary-kink Site in the CAP-DNA Complex: Recognition of Pyrimidine-Purine and Purine-Purine Steps  

SciTech Connect

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of {approx}40 deg and a twist angle of {approx}20 deg, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11-3' ('primary kink'). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an 'indirect-readout' mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T{sub 6}G{sub 7}, and the non-consensus pyrimidine-purine step C{sub 6}G{sub 7} (roll angles of {approx}42 deg, twist angles of {approx}16 deg), but is much less sharp with the non-consensus purine-purine steps A{sub 6}G{sub 7} and G{sub 6}G{sub 7} (roll angles of {approx}20 deg, twist angles of {approx}17 deg). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, {approx}46 deg per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, {approx}28 deg per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

Napoli,A.; Lawson, C.; Ebright, R.; Berman, H.

2006-01-01

153

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

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

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

2013-06-01

154

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis.  

PubMed

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I-II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E

2013-01-09

155

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

PubMed

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

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

2012-07-20

156

Comparing Study Between Four Different Methods of Genomic DNA Extraction from Cyclamen persicum Mill  

Microsoft Academic Search

The most essential principle in the modern molecular biology is extraction of DNA with a desirable quantity and quality, and this achievement and complete DNA sequencing of genome could be the main necessity for every genetic study till 2010. DNA extraction is obviously difficult because of negative effects obtained from carbohydrates, tannins, polyphenols and proteins. Therefore, the ideal method is

MITRA ALAEY; ROUHANGIZ NADERI; ALI VEZVAEI; AHMAD KHALIGHI; ALIREZA SALAMI

157

Highly Effective DNA Extraction Method from Fresh, Frozen, Dried and Clotted Blood Samples  

PubMed Central

Introduction Today, with the tremendous potential of genomics and other recent advances in science, the role of science to improve reliable DNA extraction methods is more relevant than ever before. The ideal process for genomic DNA extraction demands high quantities of pure, integral and intact genomic DNA (gDNA) from the sample with minimal co-extraction of inhibitors of downstream processes. Here, we report the development of a very rapid, less-hazardous, and high throughput protocol for extracting of high quality DNA from blood samples. Methods Dried, clotted and ethylene diamine tetra-acetic acid (EDTA) treated fresh and frozen blood samples were extracted using this method in which the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis, PCR reaction and DNA digestion using restricted enzyme. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Results PCR and DNA digestion experiments indicated that the final solutions of extracted DNA contained no inhibitory substances, which confirms that the isolated DNA is of good quality. Conclusion The high quality and quantity of current method, no enzymatic processing and accordingly its low cost, make it appropriate for DNA extraction not only from human but also from animal blood samples in any molecular biology labs.

Samadi Shams, Sara; Zununi Vahed, Sepideh; Soltanzad, Farzaneh; Kafil, Vala; Barzegari, Abolfazl; Atashpaz, Sina; Barar, Jaleh

2011-01-01

158

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

Microsoft Academic Search

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

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

2011-01-01

159

Yield and amplificability of different DNA extraction procedures for traceability in the dairy food chain  

Microsoft Academic Search

Seven DNA extraction procedures were compared for their ability to produce good quality DNA if applied to outputs of the dairy food chain. The efficacy and the efficiency of the protocols were tested. PCR and Real-time PCR specific amplification with appropriate bovine primers were used to assess the quality of the genomic DNA extracted with the compared procedures. The results

Andrea Pirondini; Urbana Bonas; Elena Maestri; Giovanna Visioli; Marta Marmiroli; Nelson Marmiroli

2010-01-01

160

Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens.  

PubMed

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat. PMID:22108495

Richardson, Leisha J; Kaestli, Mirjam; Mayo, Mark; Bowers, Jolene R; Tuanyok, Apichai; Schupp, Jim; Engelthaler, David; Wagner, David M; Keim, Paul S; Currie, Bart J

2011-11-12

161

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies.  

PubMed

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h. PMID:11280931

Santosa, D A

2001-01-01

162

Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers  

PubMed Central

Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.

Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

2012-01-01

163

Determination of chloropropanols in foods by one-step extraction and derivatization using pressurized liquid extraction and gas chromatography–mass spectrometry  

Microsoft Academic Search

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

I. Racamonde; P. González; R. A. Lorenzo; A. M. Carro

2011-01-01

164

Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin.  

PubMed Central

In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and probing the capability of chromatin to carry out repair in vitro. The rationale behind this approach is that compartmentalization of NER at nuclear structures would make the enzymatic activities refractory to extraction by buffers that solubilize cellular membranes. In order to obtain intact chromatin primary human fibroblasts were encapsulated in agarose microbeads and lysed in isotonic buffers containing the non-ionic detergent Triton X-100. Under these conditions the majority of cellular proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphates and Mg2+. UV irradiation of confluent repair-proficient human fibroblasts prior to lysis stimulated the incorporation of deoxynucleotide triphosphates in Triton X-100-isolated chromatin, even under stringent lysis conditions. In addition, experiments with UV-sensitive xeroderma pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synthesis was due to global genome repair activity. Transcription-coupled repair was only detectable in cells permeabilized by streptolysin O (SLO). Repair synthesis in Triton X-100-isolated chromatin amounted to 15% of the total repair synthesis as measured in SLO-permeabilized cells. To allow the detection of these activities in vitro, presynthesis complexes have to be formed in intact cells, indicating that chromatin from Triton X-100-lysed cells is unable to initiate NER in vitro. Our data indicate that the components involved in the resynthesis step of NER are tightly associated with chromatin. A substantial fraction of total proliferating cell nuclear antigen (PCNA), which is required for the resynthesis step in NER, has been reported to become Triton X-100 non-extractable and tightly associated with nuclear structures after UV irradiation of cells. We propose that Triton X-100-resistant repair synthesis might be mediated by this chromatin-bound fraction of total PCNA.

Bouayadi, K; van der Leer-van Hoffen, A; Balajee, A S; Natarajan, A T; van Zeeland, A A; Mullenders, L H

1997-01-01

165

A Modified Extraction Method of Circulating Free DNA for Epidermal Growth Factor Receptor Mutation Analysis  

PubMed Central

Purpose Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. Materials and Methods The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. Results MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (?202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. Conclusion An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.

Yuan, Haihua; Zhu, Zhong-Zheng; Lu, Yachao; Liu, Feng; Zhang, Wenying; Huang, Gang; Zhu, Guanshan

2012-01-01

166

Fast protocol for extraction of DNA from Prosopis spp leaves (plant adapted to arid environment) without liquid nitrogen.  

PubMed

The extraction of high-quality genomic DNA from Prosopis spp for polymerase chain reaction (PCR) amplification is complicated, owing to the presence of a high percentage of secondary metabolites that bind to or co-precipitate with nucleic acids. In the present study, we report a modified sodium dodecyl sulfate/phenol protocol that eliminates the use of liquid nitrogen in the maceration process, ?-mercaptoethanol in the buffer extraction, and the ethanol precipitation step. The A260/A280 absorbance ratios of the isolated DNA were approximately 2.0 to 1.9, suggesting that the DNA fraction was pure and can be used for further PCR analysis. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. Finally, this proposal represents an alternative fast, cheap, and effective method for the isolation of genomic DNA from fresh leaves of Prosopis spp, even in low-technology laboratories. PMID:24089098

Michel-López, C Y; González-Mendoza, D; Grimaldo-Juarez, O

2013-09-27

167

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

PubMed Central

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

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

2012-01-01

168

Comparison of different protocols for the extraction of microbial DNA from reef corals  

PubMed Central

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Santos, H.F.; Carmo, F.L.; Leite, D.C.A.; Jesus, H.E.; Maalouf, P. De Carvalho; Almeida, C.; Soriano, A.U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B.L.; Mendes, L.B.; Joao, R.R.; Lacava, B.; Jesus, R.F.; Sebastian, G.V.; Pessoa, A.; van Elsas, J.D.; Rezende, R.P.; Pires, D.O.; Duarte, G.; Castro, C.B.; Rosado, A.S.; Peixoto, R.S.

2012-01-01

169

In Xenopus Egg Extracts, DNA Replication Initiates Preferentially at or near Asymmetric AT Sequences  

Microsoft Academic Search

Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or

Slavica Stanojcic; Jean-Marc Lemaitre; Konstantin Brodolin; Etienne Danis; Marcel Mechali

2008-01-01

170

Effects of time and rainfall on PCR success using DNA extracted ...  

Treesearch

Title: Effects of time and rainfall on PCR success using DNA extracted from deer ... Source: Conservation Genetics. doi: 10.1007/s10592-009-9928-7. ... errors ( dropout and false alleles) were recorded to determine extent of DNA degradation .

171

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia  

Microsoft Academic Search

ABSTRACT. Five published DNA extraction protocols were compared for their ability to produce,good quality DNA from fresh and herbarium leaves of several,species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform,a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA

R. a. Ribeiro; M. b. Lovato

2007-01-01

172

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

PubMed

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 detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples. PMID:22629615

Zheng, Lu; Gao, Naiyun; Deng, Yang

173

Integrated Method for Single-Cell DNA Extraction, PCR Amplification, and Sequencing of Ribosomal DNA from Harmful Dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella  

Microsoft Academic Search

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and

Jang-Seu Ki; Gi Young Jang; Myung-Soo Han

2004-01-01

174

Aqueous extracts of cigarette tar containing the tar free radical cause DNA nicks in mammalian cells.  

PubMed Central

The ability of aqueous extracts of cigarette tar to nick DNA was investigated using viable mammalian cells. Tar extracts contain a radical with a stable electron spin resonance (ESR) signal at g = 2.0036 characteristic of a semiquinone. The association of the tar component that carries the ESR signal with DNA was demonstrated using viable rat alveolar macrophages. The formation of single-strand DNA breaks caused by cigarette tar extracts in viable rat thymocytes follows saturation kinetics, indicating a tar component associates with DNA and then nicks it. These studies support our hypothesis that tar components that contain the cigarette tar radical can enter cells, associate with, and then nick DNA.

Stone, K K; Bermudez, E; Pryor, W A

1994-01-01

175

Human {beta}-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old  

SciTech Connect

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

Beraud-Colomb, E. [Genetique Medicale et Developpement, Marseille (France)]|[Laboratoire d`Anthropologie, Marseille (France); Maroc, N. [Genetique Medicale et Developpement, Marseille (France); Roubin, R. [Institut Paoli-Calmettes, Marseille (France)] [and others

1995-12-01

176

A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated).  

PubMed

Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. There are many DNA extraction methods available and they vary in their ability to efficiently extract the DNA; as well as in processing time, operator intervention, contamination risk and ease of use. In recent years, automated robots have been made available which speed up processing time and decrease the amount of operator input. This project was set up to investigate the efficiency of three DNA extraction methods, two manual (Chelex(®)-100 and the QIAGEN DNA Investigator Kit) and one automated (QIAcube), using both buccal cells and blood stains as the DNA source. Extracted DNA was quantified using real-time PCR in order to assess the amount of DNA present in each sample. Selected samples were then amplified using AmpFlSTR SGM Plus amplification kit. The results suggested that there was no statistical difference between results gained for the different methods investigated, but the automated QIAcube robot made sample processing much simpler and quicker without introducing DNA contamination. PMID:21703957

Phillips, Kirsty; McCallum, Nicola; Welch, Lindsey

2011-06-23

177

Mutants of the base excision repair glycosylase, endonuclease III: DNA charge transport as a first step in lesion detection.  

PubMed

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. On the basis of 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

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

2011-06-09

178

DNA extraction from fresh-frozen and formalin-fixed, paraffinembedded human brain tissue.  

PubMed

Both fresh-frozen and formalin-fixed, paraffinembedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratorybased method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies. PMID:23996594

Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

2013-08-30

179

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

PubMed Central

Summary 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.

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

2009-01-01

180

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...

181

Rapid method for direct extraction of DNA from soil and sediments  

Microsoft Academic Search

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (> 90%), the yields of DNA were high (38 and 12

Yu-Li Tsai; B. H. Olson

1991-01-01

182

EXTRACTION OF TOTAL MICROBIAL DNA FROM THE CECEAL CONTENTS OF TURKEYS: COMPARISON OF METHODS  

Technology Transfer Automated Retrieval System (TEKTRAN)

GOAL: To identify the extraction method that gives the greatest yield and diversity of microbial DNA from the cecal contents of turkeys. METHODS: Eight commercially available kits and 14 different isolation protocols were evaluated. DNA was extracted as per the manufacturers' instructions from 0...

183

An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails  

Microsoft Academic Search

This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex® DNA extraction technique showed to be more appropriate than the classical

Y. Caron; S. Righi; L. Lempereur; C. Saegerman; B. Losson

2011-01-01

184

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts  

Microsoft Academic Search

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly

LEI LI; CAROLYN A. PETERSON; XIAOYAN LU; PING WEI; RANDY J. LEGERSKI

1999-01-01

185

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

Microsoft Academic Search

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

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

1996-01-01

186

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...

187

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents  

SciTech Connect

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. (Imperial Cancer Research Fund, South Mimms, (United Kingdom))

1991-07-01

188

Characterisation of insect and plant origins using DNA extracted from small volumes of bee honey  

Microsoft Academic Search

A DNA-based tool was validated that potentially enables the characterisation of both plant and insect of origin of small (approximately\\u000a 1 ml) samples of bee honey. Using this method, mitochondrial, nuclear and chloroplast DNA (mtDNA, nuDNA, cpDNA) markers were\\u000a successfully extracted, PCR amplified, and sequenced from a range of honeys, and the relative amount of plant nuDNA and cpDNA,\\u000a and bee

Ida Bærholm Schnell; Magdalena Fraser; Eske Willerslev; M. Thomas P. Gilbert

2010-01-01

189

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs  

Microsoft Academic Search

Summary We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconser- vative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked

J. Julian Blow; Ronald A. Laskey

1986-01-01

190

Assessing Fourth Amendment challenges to DNA extraction statutes after Samson v. California.  

PubMed

DNA plays an indispensable role in modern law enforcement, and courts uniformly find that DNA extraction statutes targeting criminals satisfy the Fourth Amendment. Courts differ on which Fourth Amendment test--totality of the circumstances or special needs--ought to be employed in this context. This Note concludes that courts should apply Samson v. California's less stringent totality of the circumstances test to analyze DNA extraction statutes in order to maintain the integrity of the special needs test. PMID:19353834

Nerko, Charles J

2008-11-01

191

Direct DNA Extraction for PCR-Mediated Assays of Soil Organisms  

Microsoft Academic Search

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

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

1995-01-01

192

EXTRACTION OF TOTAL MICROBIAL DNA FROM THE CECAL CONTENTS OF TURKEYS: COMPARISON OF METHODS  

Technology Transfer Automated Retrieval System (TEKTRAN)

GOAL: To compare turkey cecal microbial diversity present in DNA samples from eight commercially available DNA isolation kits. METHODS: Fourteen different isolation protocols from eight commercially available kits were evaluated. DNA was extracted as per the manufacturers' instructions from 0.2 ...

193

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

Microsoft Academic Search

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

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

2006-01-01

194

A continuous process to extract plasmid DNA based on alkaline lysis  

Microsoft Academic Search

Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity.

Xiaolin Li; Huali Jin; Zhifang Wu; Simon Rayner; Bin Wang

2008-01-01

195

Comparison of methods for the extraction of DNA from stream epilithic biofilms.  

PubMed

This study compares how different DNA extraction methods influence the quantity and quality of DNA yields from stream epilithic biofilms. Interpretations of bacterial community structure, using ARISA, revealed increased variability among samples processed using commercially-available kits, which also yielded lower DNA concentrations than other methods tested. PMID:20532818

Lear, Gavin; Dong, Yimin; Lewis, Gillian

2010-06-08

196

Application of a three step sequential extraction procedure for the fractionation of metals in two different types of fly ash  

Microsoft Academic Search

Two different highly homogenised fly ash materials, originating from municipal city wastes and sewage sludges, have been analysed for the total content of six elements (Cd, Cr, Cu, Ni, Pb and Zn). The distribution of these six metals among different fractions has been investigated as well, by applying a 3-step sequential extraction procedure proposed by the Standards, Measurements and Testing

C. a Brunori; Z.b Mester; C. a Cremisini; P. b Fodor; R. a Morabito

1999-01-01

197

Improving the two-step remediation process for CCA-treated wood: Part I. Evaluating oxalic acid extraction.  

PubMed

In this study, three possible improvements to a remediation process for chromated-copper-arsenate- (CCA) treated wood were evaluated. The process involves two steps: oxalic acid extraction of wood fiber followed by bacterial culture with Bacillus licheniformis CC01. The three potential improvements to the oxalic acid extraction step were (1) reusing oxalic acid for multiple extractions, (2) varying the ratio of oxalic acid to wood, and (3) using a noncommercial source of oxalic acid such as Aspergillus niger, which produces oxalic acid as a metabolic byproduct. Reusing oxalic acid for multiple extractions removed significant amounts of copper, chromium, and arsenic. Increasing the ratio of wood to acid caused a steady decline in metal removal. Aspergillus niger removed moderate amounts of copper, chromium, and arsenic from CCA-treated wood. Although A. niger was effective, culture medium costs are likely to offset any benefits. Repeated extraction with commercial oxalic acid appears to be the most cost-effective method tested for the two-step process. PMID:15081068

Clausen, Carol

2004-01-01

198

Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments.  

PubMed

PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples. PMID:16806911

Desai, Chirayu; Madamwar, Datta

2006-06-27

199

High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.  

PubMed

A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation. PMID:22841745

Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

2012-07-20

200

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

PubMed

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

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

2005-12-30

201

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

PubMed

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

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

2012-12-11

202

[The anti-dsDNA antibodies: validation of an original two step strategy of detection].  

PubMed

Detection of anti-dsDNA antibodies is one of the major biological criteria of use in the diagnosis of systemic lupus erythematosus. Sensitivity and specificity vary greatly between existing techniques, and differ largely from one study to another. The aim of our prospective study was to evaluate a new strategy of detection comprising two steps, first, the use of a sensitive automated technique, ELISA Phadia EliA™, and second, if necessary, a more specific technique: the Crithidia luciliae immunofluorescence test (CLIFT). The latter was used in case of discrepancy with previous laboratory findings or according to the available clinical data. During the study period of 18 months, 1729 tests were requested of which 96 were finally assayed using CLIFT. Analysis of 53 discordant results showed 14 cases of lupus identified only with ELISA, and 3 only by Crithidia. In addition, 35 likely false positives of ELISA were evidenced by negative CLIFT results. These data show a clear gain in sensitivity without any loss of specificity due to the use of a second technique. Thus, this strategy was validated in our lab; it can be useful by any medical laboratory because the cost of few Crithidia luciliae slides is very low. PMID:21463995

Lemarié, Romain; Jacomet, Florence; Goutte, Brigitte; Bonnafoux, Christine; Tridon, Arlette; Evrard, Bertrand

203

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

NASA Astrophysics Data System (ADS)

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 (H 2O 2). 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.

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

2011-02-01

204

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination  

Microsoft Academic Search

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations

Wen Guo; Lan Jiang; Shalender Bhasin; Shaharyar M. Khan; Russell H. Swerdlow

2009-01-01

205

Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles  

Microsoft Academic Search

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA\\/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using

Zhi Shan; Qi Wu; Xianxiang Wang; Zhongwu Zhou; Ken D. Oakes; Xu Zhang; Qianming Huang; Wanshen Yang

2010-01-01

206

Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam.  

PubMed

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis. PMID:16328977

Kim, Sun-Hyung; Hamada, Tatsuro

2005-12-01

207

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome  

Microsoft Academic Search

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of

Daniel G. Gibson; Gwynedd A. Benders; Kevin C. Axelrod; Jayshree Zaveri; Mikkel A. Algire; Monzia Moodie; Michael G. Montague; J. Craig Venter; Hamilton O. Smith; Clyde A. Hutchison

2008-01-01

208

Comparison of several methods for the extraction of DNA from potatoes and potato-derived products.  

PubMed

Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods. PMID:16366665

Smith, Donna S; Maxwell, Philip W; De Boer, Solke H

2005-12-28

209

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.  

PubMed Central

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.

Caldarelli-Stefano, R; Vago, L; Bonetto, S; Nebuloni, M; Costanzi, G

1999-01-01

210

Extracting DNA Twist Rigidity from Experimental Supercoiling Data  

NASA Astrophysics Data System (ADS)

We use an elastic rod model with contact to study the extension versus rotation diagrams of single supercoiled DNA molecules. We reproduce quantitatively the supercoiling response of overtwisted DNA and, using experimental data, we obtain an estimate of the effective supercoiling radius and of the twist rigidity of B-DNA. We find that the twist rigidity of DNA seems to vary widely with the nature and concentration of the salt buffer in which it is immersed.

Neukirch, Sébastien

2004-11-01

211

Rapid alkaline extraction method for the isolation of plasmid DNA  

Microsoft Academic Search

Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction

H. C. Birnboim

1983-01-01

212

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins  

PubMed Central

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.

Eot-Houllier, Gregory; Eon-Marchais, Severine; Gasparutto, Didier; Sage, Evelyne

2005-01-01

213

DNA extract characterization process for microbial detection methods development and validation  

PubMed Central

Background Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. Results DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/?L. Forty-seven extracts had concentrations greater than 17.5 ng/?L, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ ?L), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/?L to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/?L and 0.25 ng/?L respectively. The qPCR LOQ varied by cell type (5.72?×?10-3 ng/?L for E. coli, 2.66?×?10-3 ng/?L, for B. cereus, 3.78?×?10-3 ng/?L for B. thailandensis, and 7.67?×?10-4 ng/?L for S. cerevisiae). A number of samples were below the UV spectroscopy (n?=?27), flurometry (n?=?15), and qPCR (n?=?3) LOQ. Conclusion The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

2012-01-01

214

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis  

Microsoft Academic Search

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial

M. G. LaMontagne; F. C. Michel Jr; P. A. Holden; C. A. Reddy

2002-01-01

215

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

NASA Astrophysics Data System (ADS)

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.

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

2013-09-01

216

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples.  

PubMed

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups. PMID:22981178

Lee, Hwan Young; Yoon, Jung Ah; Yang, Woo Ick; Shin, Kyoung-Jin

2012-09-13

217

A SIMPLE VERSATILE HIGH THROUPHPUT DNA EXTRACTION METHOD SUITABLE FOR PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

PCR has become the most popular technique in functional genomics. Both forward and reverse genetic projects routinely require PCR amplification of thousands of samples. Processing the samples for DNA suitable for PCR is usually the limiting step. We have developed a simple high throughput DNA extra...

218

Twin-screw extruder for oil processing of sunflower seeds: Thermomechanical pressing and solvent extraction in a single step  

Microsoft Academic Search

A new application of twin-screw extruder as a machine to conduct a thermo-mechanical pressing and a solvent extraction of sunflower oil in a single step and in a continuous mode was studied. Experiments were conducted using a CLEXTRAL BC 45 co-rotating twin-screw extruder and whole sunflower seeds with fatty acid methyl esters as a solvent. The influences of screw rotation

I. Amalia Kartika; P. Y. Pontalier; L. Rigal

2010-01-01

219

Pulsatile culture of a poly(DL-lactic-co-glycolic acid) sandwiched cell/hydrogel construct fabricated using a step-by-step mold/extraction method.  

PubMed

To overcome the weak mechanical properties of cell/hydrogel composites, a poly(DL-lactic-co-glycolic acid) sandwiched adipose-derived stem cell (ADSC)/fibrin construct was fabricated using a step-by-step mold/extraction method to generate the middle smooth muscle layer of natural blood vessels. A pulse bioreactor with an adjustable 0-0.2 MPa pressure, 0-7% pulse amplitude, and 0-80 times/min pulse frequency was developed to mimic the liquid movement in the natural blood vessels. This new type of pulse bioreactor is sterilizable and dismantles easily. A comparative study was conducted with static and dynamic in vitro cultures. Exogenous growth factors, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor ?1, and basic fibroblast growth factor were used as additives in the culture medium for inducing the ADSCs into smooth muscle cells. The dynamic training, integrated with the growth factor, induced the transformation of ADSCs into smooth muscle-like cells with regular arrangement. This strategy shows promise of being widely used in tissue engineering and complex organ manufacturing. PMID:21671960

Wang, Xiaohong; Sui, Shaochun

2011-06-01

220

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

Microsoft Academic Search

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

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

2002-01-01

221

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

Microsoft Academic Search

Non-lethal sampling of DNA from honeybees is commonly required for genotyping certain behavioural traits required for breeding.\\u000a One method is to use wing clippings. However, the sample is very small, and the extraction process can be difficult, resulting\\u000a in low polymerase chain reaction (PCR) amplification. Here, we describe an improved method for extracting DNA from bee wings\\u000a using a commercially

Elaine M. Gould; Michelle A. Taylor; Selena J. Holmes

222

Detection of rendered meat and bone meals by PCR is dependent on animal species of origin and DNA extraction method.  

PubMed

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed. PMID:20537265

Myers, Michael J; Farrell, Dorothy E; Deaver, Christine M; Mason, Jacquline; Swaim, Heidi L; Yancy, Haile F

2010-06-01

223

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis  

PubMed Central

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Ghatak, Souvik; Muthukumaran, Rajendra Bose; Nachimuthu, Senthil Kumar

2013-01-01

224

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.  

PubMed

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass. PMID:22353211

Scaife, Mark A; Ma, Cynthia A; Armenta, Roberto E

2012-02-06

225

Assessing Fourth Amendment Challenges to DNA Extraction Statutes After Samson v. California  

Microsoft Academic Search

DNA plays an indespensable role in modern law enforcement, and courts uniformly find that DNA extraction statutes targeting criminals satisfy the Fourth Amendment. Courts differ on which Fourth Amendment test--totality of the circumstances or special needs--ought to be employed in this context. This Note concludes the courts should apply Samson v. California's less stringent totality of the circumstances test to

Charles J. Nerko

2008-01-01

226

[Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].  

PubMed

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans. PMID:22450668

Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

2012-01-01

227

One-step liquid–liquid extraction of cocaine from urine samples for gas chromatographic analysis  

Microsoft Academic Search

An improved technique for cocaine extraction from urine samples for gas chromatographic (GC) analysis is described. Employing a simple liquid–liquid extraction (LLE) of cocaine with a mixture of ethyl ether:isopropanol (9:1) the method presents a mean recovery of 74.49%. Limit of detection (LOD) and limit of quantification (LOQ) were 5 and 20ng\\/ml, respectively. The method is highly precise (coefficient of

Marcelo Farina; Maur??cio Yonamine; Ovandir A Silva

2002-01-01

228

Electron Energy Distribution Function Extraction Using Integrated Step Function Response and Regularization Methods  

Microsoft Academic Search

Recently, electron energy distribution function (EEDF) extraction techniques have been evaluated using regularized solutions to the integral problem. These techniques do not assume any mathematical representation of the EEDF and solve the integral problem for any function that best represents the EEDF. Also, unlike the more widely used point-by-point extraction of the second-derivative relationship, the integrated relationship between electron current

Ahmed El Saghir; Chris Kennedy; Steven Shannon

2010-01-01

229

Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods  

Microsoft Academic Search

Two improved DNA extraction techniques from trypan-blue-stained root fragments were developed and compared for rapid and reliable analyses. In Method A, 1 cm trypan-blue-stained mycorrhizal root fragments were individually isolated, crushed by bead beating, and purified with Chelex-100 (Bio-Rad). In Method B, DNA extraction was carried out using an UltraClean microbial DNA isolation kit (MoBio Laboratories). DNA was extracted from the

Satoshi Ishii; Thomas E. Loynachan

2004-01-01

230

A Simple Silica-based Method for Metagenomic DNA Extraction from Soil and Sediments  

Microsoft Academic Search

A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica\\u000a without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition\\u000a was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid

R. Rojas-Herrera; J. Narváez-Zapata; M. Zamudio-Maya; M. E. Mena-Martínez

2008-01-01

231

Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets  

Microsoft Academic Search

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve\\u000a problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection\\u000a method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA\\u000a degradation over time in wet environments, and

Todd J. Brinkman; Michael K. Schwartz; David K. Person; Kristine L. Pilgrim; Kris J. Hundertmark

2010-01-01

232

Fabrication of amino silane-coated microchip for DNA extraction from whole blood  

Microsoft Academic Search

A simple microchip device for DNA extraction was constructed based on electrostatic interactions between surface amine groups and DNA. Microchannel was fabricated on silicon wafer by photolithography and coated with 3-aminopropyltriethoxysilane (APTES) or 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to introduce amine groups on the surface. Determination of the number of surface amine groups and optimization of DNA capture condition were demonstrated to characterize

Takahito Nakagawa; Tsuyoshi Tanaka; Daisuke Niwa; Tetsuya Osaka; Haruko Takeyama; Tadashi Matsunaga

2005-01-01

233

Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor  

Microsoft Academic Search

In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the OH radicals via Fenton-type reaction. The interaction

Lucilene Dornelles Mello; Silvia Hernandez; Giovanna Marrazza; Marco Mascini; Lauro Tatsuo Kubota

2006-01-01

234

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy  

Microsoft Academic Search

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluores- cence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spec- troscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by invest- igating bimolecular quenching

Jiho Kim; Soren Doose; Hannes Neuweiler; Markus Sauer

2006-01-01

235

Increased Aortic DNA Synthesis Precedes Renal Hypertension in Rats An Obligatory Step?  

Microsoft Academic Search

3 H)thymidine incorpor- ation into DNA per hour. After stenosis of the renal artery, blood pressure increased over a 2-week period. Five days after clipping, there was an increase in the rate of aortic DNA synthesis before an increase in blood pressure was detected, whereas there was no DNA effect in sham-operated animals. This difference in ( 3 H)thymidine incorporation

ALEX L. LOEB; H. GEORGE MANDEL; JAMES A. STRAW; BARBARA L. BEAN

236

Highly efficient factorial designs for cDNA microarray experiments: use of approximate theory together with a step-up step-down procedure.  

PubMed

Abstract A general method for obtaining highly efficient factorial designs of relatively small sizes is developed for cDNA microarray experiments. It allows the main effects and interactions to be of possibly unequal importance. First, the approximate theory is employed to get an optimal design measure which is then discretized. It is, however, observed that a naïve discretization may fail to yield an exact design of the stipulated size and, even when it yields such an exact design, there is often scope for improvement in efficiency. To address these issues, we propose a step-up/down procedure which is seen to work very well. The resulting designs turn out to be quite robust to possible dye-color effects and heteroscedasticity. We focus on the baseline and all-to-next parametrizations but our method works equally well also for hybrids of the two and other parametrizations. PMID:23813301

Zhang, Runchu; Mukerjee, Rahul

2013-08-01

237

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

PubMed

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

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

2012-04-01

238

Terminal DNA structure and ATP influence binding parameters of the DNA-dependent protein kinase at an early step prior to DNA synapsis  

PubMed Central

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the non-homologous end-joining pathway of DNA double-strand break repair in mammalian cells. The ability of DNA-PKcs to sense and respond to different terminal DNA structures is postulated to be important for its regulatory function. It is unclear whether discrimination occurs at the time of formation of the initial protein–DNA complex or later, at the time of formation of a paired, or synaptic complex between opposing DNA ends. To gain further insight into the mechanism of regulation, we characterized the binding of DNA-PKcs to immobilized DNA fragments that cannot undergo synapsis. Results showed that DNA-PKcs strongly discriminates between different terminal structures at the time of initial complex formation. Although Ku protein stabilizes DNA-PKcs binding overall, it is not required for discrimination between terminal structures. Base mispairing, temperature and the presence of an interstrand linkage influence the stability of the initial complex in a manner that suggests a requirement for DNA unwinding, reminiscent of the ‘open complex’ model of RNA polymerase–promoter DNA interaction. ATP and a nonhydrolyzable ATP analog also influence the stability of the DNA-PKcs•DNA complex, apparently by an allosteric mechanism that does not require DNA-PKcs autophosphorylation.

Jovanovic, Marko; Dynan, William S.

2006-01-01

239

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

Microsoft Academic Search

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

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

1999-01-01

240

Microwave assisted extraction combined with solvent bar microextraction for one-step solvent-minimized extraction, cleanup and preconcentration of polycyclic aromatic hydrocarbons in soil samples.  

PubMed

For the first time, a novel one-step sample preparation method that combines microwave assisted extraction and solvent bar microextraction (MAE-SBME) with analysis by gas chromatography-mass spectrometry (GC-MS), was developed for the fast and efficient determination of polycyclic aromatic hydrocarbons (PAHs) in environmental soil samples. An interesting feature of the new procedure is that SBME was conducted simultaneously with MAE. Thus, the extract from the SBME could be directly and immediately analyzed by GC-MS. A separate clean up and/or preconcentration process, such as time-consuming and tedious gel permeation chromatography, solid-phase extraction, filtration, or adsorption chromatography, normally associated with conventional MAE, was not necessary. It is also notable that the procedure was environmentally benign since water was used as the extraction solvent in MAE, and only several microliters of organic solvent were used in SBME. Some factors affecting the extraction were studied and optimized. Under the most favorable conditions, the method showed good linearities (between 0.2 and 500, 0.5 and 500, 1 and 500, and 2 and 500 ng/g, depending on the analytes), low limits of detection (from 0.03 to 0.25 ng/g), and satisfactory precision (with relative standard deviations below 9.8%). The MAE-SBME procedure provides a fast and simple sample preparation approach for the processing of environmental soil samples. PMID:23497848

Guo, Liang; Lee, Hian Kee

2013-02-27

241

Conformational characteristics of DNA: empirical classifications and a hypothesis for the conformational behaviour of dinucleotide steps  

Microsoft Academic Search

This paper is concerned with an investigation of the geometry and structure of DNA as revealed by X-ray diffraction of single crystal oligomeric structures. A database of atomic coordinates of 60 naked (i.e. not bound to any protein or drug) DNA oligomers (25 dodecamers, 18 decamers, 16 octamers and 1 tetramer) is set up and carefully described. An extensive empirical

M. A. El Hassan; C. R. Calladine

1997-01-01

242

Conformational Characteristics of DNA: Empirical Classifications and a Hypothesis for the Conformational Behaviour of Dinucleotide Steps  

Microsoft Academic Search

This paper is concerned with an investigation of the geometry and structure of DNA as revealed by X-ray diffraction of single crystal oligomeric structures. A database of atomic coordinates of 60 naked (i.e. not bound to any protein or drug) DNA oligomers (25 dodecamers, 18 decamers, 16 octamers and 1 tetramer) is set up and carefully described. An extensive empirical

M. A. El Hassan; C. R. Calladine

1997-01-01

243

Two-Step Recruitment of RNA-Directed DNA Methylation to Tandem Repeats  

Microsoft Academic Search

Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence

Simon W.-L. Chan; Xiaoyu Zhang; Yana V. Bernatavichute; Steven E. Jacobsen

2006-01-01

244

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.  

PubMed Central

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Aljanabi, S M; Martinez, I

1997-01-01

245

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts  

PubMed Central

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.

Li, Lei; Peterson, Carolyn A.; Lu, Xiaoyan; Wei, Ping; Legerski, Randy J.

1999-01-01

246

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue.  

PubMed

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies. PMID:22449494

Chung, Joon-Yong; Yi, Joo Mi; Xie, Ran; Brown, Victoria; Lee, Olivia; Ahuja, Nita; Braunschweig, Till; Hewitt, Stephen M

2012-03-23

247

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis.  

PubMed

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 ?g ?L(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost. PMID:23239373

Tian, Wei; Zhang, Zhenhua; Liu, Dongyang; Zhou, Tiantian; Shen, Qirong; Shen, Biao

2012-12-14

248

First-passage problems in DNA replication: effects of template tension on stepping and exonuclease activities of a DNA polymerase motor.  

PubMed

A DNA polymerase (DNAP) replicates a template DNA strand. It also exploits the template as the track for its own motor-like mechanical movement. In the polymerase mode it elongates the nascent DNA by one nucleotide in each step. However, whenever it commits an error by misincorporating an incorrect nucleotide, it can switch to an exonuclease mode. In the latter mode it excises the wrong nucleotide before switching back to its polymerase mode. We develop a stochastic kinetic model of DNA replication that mimics an in vitro experiment where single-stranded DNA, subjected to a mechanical tension F, is converted to double-stranded DNA by a single DNAP. The F-dependence of the average rate of replication, which depends on the rates of both polymerase and exonuclease activities of the DNAP, is in good qualitative agreement with the corresponding experimental results. We introduce nine novel distinct conditional dwell times of a DNAP. Using the method of first-passage times, we also derive the exact analytical expressions for the probability distributions of these conditional dwell times. The predicted F-dependences of these distributions are, in principle, accessible to single-molecule experiments. PMID:23945294

Sharma, Ajeet K; Chowdhury, Debashish

2013-08-15

249

First-passage problems in DNA replication: effects of template tension on stepping and exonuclease activities of a DNA polymerase motor  

NASA Astrophysics Data System (ADS)

A DNA polymerase (DNAP) replicates a template DNA strand. It also exploits the template as the track for its own motor-like mechanical movement. In the polymerase mode it elongates the nascent DNA by one nucleotide in each step. However, whenever it commits an error by misincorporating an incorrect nucleotide, it can switch to an exonuclease mode. In the latter mode it excises the wrong nucleotide before switching back to its polymerase mode. We develop a stochastic kinetic model of DNA replication that mimics an in vitro experiment where single-stranded DNA, subjected to a mechanical tension F, is converted to double-stranded DNA by a single DNAP. The F-dependence of the average rate of replication, which depends on the rates of both polymerase and exonuclease activities of the DNAP, is in good qualitative agreement with the corresponding experimental results. We introduce nine novel distinct conditional dwell times of a DNAP. Using the method of first-passage times, we also derive the exact analytical expressions for the probability distributions of these conditional dwell times. The predicted F-dependences of these distributions are, in principle, accessible to single-molecule experiments.

Sharma, Ajeet K.; Chowdhury, Debashish

2013-09-01

250

A hyper-step DNA computing system based on surface plasmon resonance  

NASA Astrophysics Data System (ADS)

We reported a reusable DNA computing platform for solving satisfiability (SAT) problem based on surface plamon resonance (SPR) technology in this paper. Three different sequences of 18-mer ssDNAs with thiol terminal were first immobilized on the gold surface and then hybridized with their complementary sequences at specific sites via microfluidic channels under room temperature. We also conjugated monoclonal antibody (human IgG) to these complementary pairs chemically to amplify the hybridization signal and thus enhance the noise margin to distinguish Boolean value of true and false. In order to keep the reaction temperature and SPR measurement stable, repeated DNA annealing and denaturing is doned by varying salt concentration (by adding NaOH to denature DNA) of reaction solution rather than changing reaction temperature. The experimental results successfully demonstrated a multi-channel microfluidic DNA computation system to solve a three variables (X, Y, Z) Boolean SAT problem (formula) with reusability and specificity using protein-ssDNA conjugates to link to complementary ssDNA SAM surface under room temperature within one hour. This technique provide a feasible solution to miniaturize the DNA computation platform for possible iterated hyperstep computing processes.

Chang, Tsung-Yao; Lin, Che-Hsin; Yang, Chia-Ning; Lin, Chii-Wann

2007-01-01

251

A practical and novel method to extract genomic DNA from blood collection kits for plasma protein preservation.  

PubMed

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources. The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes(1), to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes(2-4). Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel. We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol. PMID:23711730

Waters, Jon; Dhere, Vishal; Benjamin, Adam; Sekar, Arvind; Kumar, Archana; Prahalad, Sampath; Okou, David T; Kugathasan, Subra

2013-05-18

252

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

PubMed Central

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.

Parrilla-Doblas, Jara Teresa; Ponferrada-Marin, Maria Isabel; Roldan-Arjona, Teresa; Ariza, Rafael R.

2013-01-01

253

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

PubMed

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

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

2013-07-18

254

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.  

PubMed

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods. PMID:16621089

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-04-18

255

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

PubMed Central

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.

2010-01-01

256

Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract.  

PubMed

The Xenopus early embryonic cell cycle consists of rapid oscillations between mitosis and DNA synthesis. We used ubiquitin (Ub)-dependent proteolysis inhibitors to determine whether Ub-mediated proteolysis regulates the initiation of DNA replication in Xenopus egg extract. Methylated Ub, a chemically modified Ub that cannot form chains, and S5a, a Ub chain-binding subunit of the 26S proteasome, were added to extract at concentrations known to inhibit cyclin B proteolysis and their effects on cell cycle progression and DNA replication were examined. DNA replication initiated concomitant with controls and proceeded in a semiconservative fashion in the presence of both methylated Ub and S5a. However, mitotic progression was halted, showing that the inhibitors were functional. We conclude that initiation of DNA replication is not regulated by Ub-dependent proteolysis in the early Xenopus cell cycle. PMID:12915114

Mahaffey, David T; Gorbea, Carlos; Rechsteiner, Martin

2003-08-15

257

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.  

PubMed

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. PMID:1935910

Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

1991-12-01

258

A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates.  

PubMed

Direct PCR generates DNA profiles from samples without using the extraction process. During sample extraction, DNA may be lost due to the methods used, which can affect the quality of the DNA profile obtained. This is not the case with direct PCR, where the sample is transferred directly into the PCR tube. Here, we report on the ability of direct PCR to generate DNA profiles from low amounts of control DNA retrieved from various surfaces using PowerPlex 16 HS. A comparison is made with samples undergoing a preliminary extraction stage using QiaAmp DNA Micro kits. Samples subjected to direct PCR generated DNA profiles with higher peak heights and lower allele dropout on all the different substrates tested when compared to the samples subjected to extraction. The amount of DNA retrieved from each substrate also varied even though the same amount of starting material was deposited, proving that the type of substrate can affect the retrieval of DNA. PMID:21925992

Swaran, Yuvaneswari Chandramoulee; Welch, Lindsey

2011-09-16

259

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells  

PubMed Central

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.

Fourcade, A.; Huynh, T.; Lacour, Fanny

1974-01-01

260

Automated DNA Extraction, Quantification, Dilution, and PCR Preparation for Genotyping by High-Resolution Melting  

PubMed Central

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (Tm). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on Tm, as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.

Seipp, Michael T.; Herrmann, Mark; Wittwer, Carl T.

2010-01-01

261

Reactive molecular dynamics study on the first steps of DNA damage by free hydroxyl radicals.  

PubMed

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 propagates to a DNA single and double strand break. PMID:21882859

Abolfath, Ramin M; van Duin, A C T; Brabec, Thomas

2011-09-19

262

Single-step rapid assembly of DNA origami nanostructures for addressable nanoscale bioreactors.  

PubMed

Self-assembled DNA origami nanostructures have shown great promise for bottom-up construction of complex objects with nanoscale addressability. Here we show that DNA origami-based 1D nanoribbons and nanotubes are one-pot assembled with controllable sizes and nanoscale addressability with high speed (within only 10-20 min), exhibiting extraordinarily high cooperativity that is often observed in assembly of natural molecular machines in cells (e.g. ribosome). By exploiting the high specificity of DNA-based self-assembly, we can precisely anchor proteins on these DNA origami nanostructures with sub-10 nm resolution and at the single-molecule level. We attach a pair of enzymes (horseradish peroxidase and glucose oxidase) at the inner side of DNA nanotubes and observe high coupling efficiency of enzyme cascade within this confined nanospace. Hence, DNA nanostructures with such unprecedented properties shed new light on the design of nanoscale bioreactors and nanomedicine and provide an artificial system for studying enzyme activities and cascade in highly organized and crowded cell-mimicking environments. PMID:23237536

Fu, Yanming; Zeng, Dongdong; Chao, Jie; Jin, Yanqiu; Zhang, Zhao; Liu, Huajie; Li, Di; Ma, Hongwei; Huang, Qing; Gothelf, Kurt V; Fan, Chunhai

2012-12-28

263

Radiostrontium separation and measurement in a single step using plastic scintillators plus selective extractants. Application to aqueous sample analysis.  

PubMed

This study describes a new protocol for (90)Sr determination in water samples based on the use of a selective extractant (DtBuCH18C6) and plastic scintillator microspheres. The proposed procedure unifies chemical separation and sample measurement preparation in a single step to reduce the effort, time and reagents required for analysis. In addition, the final measurement does not produce mixed waste. The minimum activity detectable for 10 mL of sample solution is 0.46 Bq L(-1). Relative errors for the determination of (90)Sr activity in drinking, sea and river waters are less than 4%. PMID:21237307

Bagán, H; Tarancón, A; Rauret, G; García, J F

2010-12-01

264

Green synthesis of Au–Pd bimetallic nanoparticles: Single-step bioreduction method with plant extract  

Microsoft Academic Search

A facile and eco-friendly method for the preparation of Au–Pd bimetallic nanoparticles (~7nm) has been developed based on simultaneous bioreduction of Au(III) and Pd(II) precursors with Cacumen Platycladi leaf extract in aqueous environment. The morphology, structure, and size were confirmed with the aid of transmission electron microscopy, selected area electron diffraction, UV–vis spectroscopy, X-ray diffraction, and energy dispersive X-ray spectroscopy.

Guowu Zhan; Jiale Huang; Mingming Du; Ibrahim Abdul-Rauf; Yao Ma; Qingbiao Li

2011-01-01

265

Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR.  

PubMed

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood. PMID:18287291

Metwally, L; Fairley, D J; Coyle, P V; Hay, R J; Hedderwick, S; McCloskey, B; O'Neill, H J; Webb, C H; Elbaz, W; McMullan, R

2008-03-01

266

Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR  

NASA Astrophysics Data System (ADS)

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.

Ansari, Aseem Z.; Chael, Mark L.; O'Halloran, Thomas V.

1992-01-01

267

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins  

PubMed Central

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.

Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian

2012-01-01

268

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.  

PubMed Central

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference. Images

Jessberger, R; Heuss, D; Doerfler, W

1989-01-01

269

Quality of DNA extracted from saliva samples collected with the Oragene(TM) DNA self-collection kit  

PubMed Central

Background Large epidemiological studies in DNA biobanks have increasingly used less invasive methods for obtaining DNA samples, such as saliva collection. Although lower amounts of DNA are obtained as compared with blood collection, this method has been widely used because of its more simple logistics and increased response rate. The present study aimed to verify whether a storage time of 8?months decreases the quality of DNA from collected samples. Methods Saliva samples were collected with an OrageneTM DNA Self-Collection Kit from 4,110 subjects aged 14–15?years. The samples were processed in two aliquots with an 8-month interval between them. Quantitative and qualitative evaluations were carried out in 20% of the samples by spectrophotometry and genotyping. Descriptive analyses and paired t-tests were performed. Results The mean volume of saliva collected was 2.2?mL per subject, yielding on average 184.8??g DNA per kit. Most samples showed a Ratio of OD differences (RAT) between 1.6 and 1.8 in the qualitative evaluation. The evaluation of DNA quality by TaqMan®, High Resolution Melting (HRM), and restriction fragment length polymorphism-PCR (RFLP-PCR) showed a rate of success of up to 98% of the samples. The sample store time did not reduce either the quantity or quality of DNA extracted with the Oragene kit. Conclusion The study results showed that a storage period of 8?months at room temperature did not reduce the quality of the DNA obtained. In addition, the use of the Oragene kit during fieldwork in large population-based studies allows for DNA of high quantity and high quality.

2012-01-01

270

PicoNewton-Millisecond Force Steps Reveal the Transition Kinetics and Mechanism of the Double-Stranded DNA Elongation  

PubMed Central

We study the kinetics of the overstretching transition in ?-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5–2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ?25 basepairs. This mechanism increases the free energy for the elementary reaction to ?94 kBT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.

Bianco, Pasquale; Bongini, Lorenzo; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo

2011-01-01

271

Enhanced extraction of heavy metals in the two-step process with the mixed culture of Lactobacillus bulgaricus and Streptococcus thermophilus.  

PubMed

For biological extraction of heavy metals from chromated copper arsenate (CCA) treated wood, different bacteria were investigated. The extraction rates of heavy metals using Lactobacillusbulgaricus and Streptococcusthermophilus were highest. The chemical extraction rates were depended on the amounts of pyruvic acid and lactic acid. Especially, the extraction rates using mixed pyruvic acid and lactic acid were increased compared to those of sole one. They were also enhanced in the mixed culture of L. bulgaricus and S. thermophilus. To improve the extraction of CCA, a two-step processing procedure with the mixed culture of L. bulgaricus and S. thermophilus was conducted. A maximum of 93% of copper, 86.5% of chromium, and 97.8% of arsenic were extracted after 4 days. These results suggest that a two-step process with the mixed culture of L. bulgaricus and S. thermophilus is most effective to extract heavy metals from CCA treated wood. PMID:22019399

Chang, Young-Cheol; Choi, DuBok; Kikuchi, Shintaro

2011-09-20

272

Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores  

SciTech Connect

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.

Torok, Tamas

2003-05-19

273

Determination of oil reservoir radiotracer (S14CN-) in a single step using a plastic scintillator extractive resin.  

PubMed

The analysis of radiotracers is important in the study of oil reservoir dynamics. One of the most widely used radiotracer is S(14)CN(-). Prior to activity measurements by Liquid Scintillation (LS), routine determinations require the pretreatment steps of purification and concentration of the samples using anion exchange columns. The final elution media produces samples with high salt concentration that may lead to problems with phase separation during the LS measurement. Plastic Scintillation (PS) is an alternative technique that provides a solid surface that can be used as a platform for the immobilisation of selective extractants to obtain a PS resin. The proposed procedure unifies chemical separation and sample measurement preparation in a single step, serving to reduce the number of reagents needed and manpower required for the analysis while also avoiding mixed waste production by LS. The objective of this study is to develop a PS resin for the determination of (14)C-labelled thiocyanate radiotracer in water samples. For this purpose, the immobilisation procedure was optimised, including optimisation of the proportion of PS microspheres:extractant and the use of a control blank to monitor the PS resin immobilisation process. The breakthrough volume was studied and the detection and quantification limits for 100 mL of sample were determined to be 0.08 Bq L(-1) and 0.31 Bq L(-1), respectively. The established procedure was applied to active samples from oil reservoirs and errors lower than 5% in the sample determinations were obtained. PMID:22769002

Bagán, H; Tarancón, A; Stavsetra, L; Rauret, G; García, J F

2012-05-31

274

Application of FTA technology to extraction of sperm DNA from mixed body fluids containing semen.  

PubMed

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA card is available for application to routine work. PMID:16221558

Fujita, Yoshihiko; Kubo, Shin-ichi

2005-10-10

275

Protection from radiation-induced mitochondrial and genomic DNA damage by an extract of Hippophae rhamnoides.  

PubMed

Hippophae rhamnoides or seabuckthorn is used extensively in Indian and Tibetan traditional medicine for the treatment of circulatory disorders, ischemic heart disease, hepatic injury, and neoplasia. In the present study, we have evaluated the radioprotective potential of REC-1001, a fraction isolated from the berries of H. rhamnoides. Chemical analysis of the extract indicated that REC-1001 was approximately 68% by weight polyphenols, and contained kaempferol, isorhamnetin, and quercetin. The effect of REC-1001 on modulating radiation-induced DNA damage was determined in murine thymocytes by measuring nonspecific nuclear DNA damage at the whole genome level using the alkaline halo assay and by measuring sequence/gene-specific DNA damage both in nuclear DNA (beta-globin gene) and in mitochondrial DNA using a quantitative polymerase chain reaction. Treatment with 10 Gy resulted in a significant amount of DNA damage in the halo assay and reductions in the amplification of both the beta-globin gene and mitochondrial DNA. REC-1001 dose-dependently reduced the amount of damage detected in each assay, with the maximum protective effects observed at the highest REC-1001 dose evaluated (250 micro g/ml). Studies measuring the nicking of naked plasmid DNA further established the radioprotective effect of REC-1001. To elucidate possible mechanisms of action, the antioxidant properties and the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependently scavenged radiation-induced hydroxyl radicals, chemically-generated superoxide anions, stabilized DPPH radicals, and reduced Fe(3+) to Fe(2+). The results of the study indicate that the REC-1001 extract of H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage. The polyphenols/flavonoids present in the extract might be responsible for the free radical scavenging and DNA protection afforded by REC-1001. PMID:16948057

Shukla, Sandeep Kumar; Chaudhary, Pankaj; Kumar, Indracanti Prem; Samanta, Namita; Afrin, Farhat; Gupta, Manju Lata; Sharma, Upendra Kumar; Sinha, Arun Kumar; Sharma, Yogendra Kumar; Sharma, Rakesh Kumar

2006-12-01

276

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

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

Yazdinezhad, Samaneh Raouf; Ballesteros-Gómez, Ana; Lunar, Loreto; Rubio, Soledad

2013-04-01

277

A high throughput DNA extraction method with high yield and quality  

PubMed Central

Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

2012-01-01

278

An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction.  

PubMed

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction. PMID:23838266

Imaizumi, Kazuhiko; Taniguchi, Kei; Ogawa, Yoshinori

2013-07-06

279

Highly Effective DNA Extraction Method for Nuclear Short Tandem Repeat Testing of Skeletal Remains from Mass Graves  

PubMed Central

Aim To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves. Methods DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit. Results DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles. Conclusions The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.

Davoren, Jon; Vanek, Daniel; Konjhodzic, Rijad; Crews, John; Huffine, Edwin; Parsons, Thomas J.

2007-01-01

280

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts  

Microsoft Academic Search

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial\\u000a DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples\\u000a were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality\\u000a samples was

Nathan R. Campbell; Shawn R. Narum

2009-01-01

281

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

Microsoft Academic Search

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

Scott O. Rogers; Arnold J. Bendich

1985-01-01

282

Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex  

PubMed Central

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a “capture probe” attached by its 5? terminus to a gold electrode and a 5? methylene blue-modified “signaling probe” that is complementary at both its 3? and 5? termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5? end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Xiao, Yi; Lubin, Arica A.; Baker, Brian R.; Plaxco, Kevin W.; Heeger, Alan J.

2006-01-01

283

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.  

PubMed

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria. PMID:2403255

Welch, D; Lee, C H; Larsen, S H

1990-08-01

284

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)  

PubMed Central

We describe a format for production of protein arrays termed ‘protein in situ array’ (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, VH/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

He, Mingyue; Taussig, Michael J.

2001-01-01

285

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

PubMed

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

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

2013-03-13

286

[Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR].  

PubMed

Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR. PMID:18368754

Zinchenko, O V; Antonov, V A; Tkachenko, G A; Altukhova, V V; Zamaraev, V S; Piven', N N; Goloseev, Iu A; Vasil'ev, V P; Lomova, L V; Alekseev, V V

287

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

Microsoft Academic Search

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

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

2004-01-01

288

Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay.  

PubMed

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 human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2. PMID:15630179

Park, Yoo Kyoung; Lee, Hyang Burm; Jeon, Eun-Jae; Jung, Hack Sung; Kang, Myung-Hee

2004-01-01

289

An alternative for the extraction and storage of DNA from insects in forensic entomology.  

PubMed

An important area of recent research in forensic entomology has been the use of insect DNA to provide identification of insects for fast and accurate estimation of time since death. This requires DNA to be extracted efficiently and in a state suitable for use in molecular procedures, and then stored on a long-term basis. In this study, Whatman FTA cards were tested for use with the Calliphoridae (Diptera). In particular, testing examined their ability to effectively extract DNA from specimens, and store and provide DNA template in a suitable condition for amplification using the polymerase chain reaction (PCR). The cards provided DNA that was able to be amplified from a variety of life stages, and thus appears to be of sufficient quality and quantity for use in subsequent procedures. FTA cards therefore appear suitable for use with calliphorids, and provide a new method of extraction that is simple and efficient and allows for storage and transportation without refrigeration, consequently simplifying the handling of DNA in forensic entomological cases. PMID:15932097

Harvey, Michelle L

2005-05-01

290

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

Microsoft Academic Search

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

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

1997-01-01

291

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

NASA Astrophysics Data System (ADS)

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.

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

2009-05-01

292

Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars  

Microsoft Academic Search

Direct genomic DNA extraction from vinegars was set up and suitability for PCR assays performed by PCR\\/DGGE and sequencing of 16S rRNA gene. The method was tested on 12 intermediary products of special vinegars, fruit vinegars and condiments produced from different raw materials and procedures. DNAs extraction was performed on pellets by chemical, enzymatic, resin mediated methods and their modifications.

Dhouha Mamlouk; Claudio Hidalgo; María-Jesús Torija; Maria Gullo

2011-01-01

293

Inhibitory activities of microalgal extracts against Epstein-Barr virus DNA release from lymphoblastoid cells  

Microsoft Academic Search

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\\u000a activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The\\u000a methanol extracts from Ankistrodesmus

Yih-Yih Kok; Wan-Loy Chu; Siew-Moi Phang; Shar Mariam Mohamed; Rakesh Naidu; Pey-Jiun Lai; Shui-Nyuk Ling; Joon-Wah Mak; Patricia Kim-Chooi Lim; Pauline Balraj; Alan Soo-Beng Khoo

2011-01-01

294

Cascade cell lyses and DNA extraction for identification of genes and microorganisms in kefir grains.  

PubMed

Kefir is a dairy product popular in many countries in Central Europe, especially in Poland and other countries of Eastern and Northern Europe. This type of fermented milk is produced by a complex population of symbiotic bacteria and yeasts. In this work, conditions for DNA extraction, involving disruption of kefir grains and a cascade of cell lysis treatments, were established. Extraction procedure of total microbial DNA was carried out directly from fresh kefir grains. Using different lysis stringency conditions, five DNA pools were obtained. Genetic diversity of DNA pools were validated by RAPD analysis, which showed differences in patterns of amplified DNA fragments, indicating diverse microbial composition of all the analysed samples. These DNA pools were used for construction of genomic DNA libraries for sequencing. As much as 50% of the analysed nucleotide sequences showed homology to sequences from bacteria belonging to the Lactobacillus genus. Several sequences were similar to sequences from bacteria representing Lactococcus, Oenococcus, Pediococcus, Streptococcus and Leuconostoc species. Among homologues of yeast proteins were those from Candida albicans, Candida glabrata, Kluyveromyces lactis and Saccharomyces cerevisiae. In addition, several sequences were found to be homologous to sequences from bacteriophages. PMID:22008413

Kowalczyk, Magdalena; Kolakowski, Piotr; Radziwill-Bienkowska, Joanna M; Szmytkowska, Agnieszka; Bardowski, Jacek

2011-10-13

295

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

PubMed Central

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.

2012-01-01

296

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication  

PubMed Central

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase ?-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

Weisshart, Klaus; Taneja, Poonam; Fanning, Ellen

1998-01-01

297

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  

Microsoft Academic Search

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

Surjit B. Dixit; David L. Beveridge; David A. Case; Thomas E. Cheatham; Emmanuel Giudice; Filip Lankas; Richard Lavery; John H. Maddocks; Roman Osman; Heinz Sklenar; Kelly M. Thayer; Péter Varnai

2005-01-01

298

Chaga mushroom extract inhibits oxidative DNA damage in lymphocytes of patients with inflammatory bowel disease.  

PubMed

Inflammatory Bowel Disease (IBD) is partly caused by oxidative stress from free radicals and reduced antioxidant levels. Using hydrogen peroxide to induce oxidative stress in vitro in peripheral lymphocytes we investigated the induction of DNA damage supplemented with ethanolic extract of Chaga mushroom as a protective antioxidant. Lymphocytes were obtained from 20 IBD patients and 20 healthy volunteers. For treatment, a constant H_{2}O_{2 } dose (50 microg/ml) was used with variable doses of Chaga extract (10-500 microg/ml). DNA damage was evaluated in 50 cells per individual and dose using the Comet assay (making 1000 observations per experimental point ensuring appropriate statistical power). Chaga supplementation resulted in a 54.9% (p < 0.001) reduction of H_{2}O_{2 } induced DNA damage within the patient group and 34.9% (p < 0.001) within the control group. Lymphocytes from Crohn's disease (CD) patients had a greater basic DNA damage than Ulcerative Colitis (UC) patients (p < 0.001). Conclusively, Chaga extract reduces oxidative stress in lymphocytes from IBD patients and also healthy individuals when challenged in vitro. Thus, Chaga extract could be a possible and valuable supplement to inhibit oxidative stress in general. PMID:18997282

Najafzadeh, Mojgan; Reynolds, P Dominic; Baumgartner, Adolf; Jerwood, David; Anderson, Diana

2007-01-01

299

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?  

PubMed

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants. PMID:15247494

Vuosku, Jaana; Jaakola, Laura; Jokipii, Soile; Karppinen, Katja; Kämäräinen, Terttu; Pelkonen, Veli-Pekka; Jokela, Anne; Sarjala, Tytti; Hohtola, Anja; Häggman, Hely

2004-07-01

300

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

Microsoft Academic Search

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

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

1997-01-01

301

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

Microsoft Academic Search

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

Ibrahim Abdullah Alaraidh

302

Robotic DNA extraction system as a new way to process sweat traces rapidly and efficiently  

Microsoft Academic Search

A modified DNA IQ™ System (Promega Corporation, USA) was used on a variety of exhibits collected at different crime scenes, potentially interested by sweat traces and analysed by means of a fully automated extraction (MultiProbe II Plus EX by Perkin Elmer LAS, USA). As is well known, sweat can be soaked into large portions of fabrics and clothes; as a

M. Pizzamiglio; A. Marino; G. Portera; D. My; C. Bellino; L. Garofano

2006-01-01

303

Easy DNA extraction for rapid detection of Panax ginseng C. A. Meyer in commercial ginseng products  

Microsoft Academic Search

An investigation was carried out in order to obtain an easy and rapid detection of Panax ginseng in commercial herbal products by using molecular techniques (polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)). Two protocols and one commercial kit for DNA extraction were used. Four forms of commercial products were considered, i.e., dried body roots, dried root tails,

P. Del Serrone; L. Attorri; G. Palazzino

2007-01-01

304

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

Microsoft Academic Search

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

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

2007-01-01

305

DNA degradation by aqueous extract of Aloe vera in the presence of copper ions.  

PubMed

The plant Aloe vera has long been used in medicine, as dietary supplements and for cosmetic purposes. Aloe vera extracts are a rich source of polyphenols, such as aloin and aloe emodin and have shown a wide range of pharmacological properties, including anti-inflammatory and anti-cancer properties. The bioactive component aloe emodin has been reported to induce apoptosis in various cancer cell lines. Many of the biological activities of Aloe vera have been attributed to its antioxidant properties. However, most plant-derived polyphenols that are also present in Aloe vera may exhibit pro-oxidant properties either alone or in the presence of transition metals, such as copper. Previous reports from this laboratory have implicated the pro-oxidant action as one of the mechanisms for their anti-cancer properties. In the present paper, we show that aqueous extract of Aloe vera is also able to cause DNA degradation in the presence of copper ions. Further, the extract is also able to reduce Cu(II) to Cu(I) and generate reactive oxygen species, such as superoxide anion and hydroxyl radicals in a dose-dependent manner, which correlates with ability of the extract to cause DNA breakage. Thus, the study shows that in addition to antioxidant activity, Aloe vera extract also possess pro-oxidant properties, leading to oxidative DNA breakage. PMID:20653287

Naqvi, Shoa; Ullah, M F; Hadi, S M

2010-06-01

306

Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.  

PubMed Central

Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk of the RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology. Images

Zaitlin, D; Hu, J; Bogorad, L

1989-01-01

307

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols.  

PubMed

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1×10(6) cells/cm(3). This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals. PMID:22005039

Alain, Karine; Callac, Nolwenn; Ciobanu, Maria-Cristina; Reynaud, Yann; Duthoit, Frédérique; Jebbar, Mohamed

2011-10-07

308

Observing dynamics of chromatin fibers in Xenopus egg extracts by single DNA manipulation using a transverse magnetic tweezer setup  

Microsoft Academic Search

We have studied assembly of chromatin on single DNAs using Xenopus egg extracts and a specially designed magnetic tweezer setup which generates controlled force in the focal plane of the objective, allowing us to visualize and measure DNA extension under a wide range of constant tensions. We found, in the absence of ATP, interphase extracts assembled nucleosomes against DNA tensions

Jie Yan; Dunja Skoko; John Marko; Tom Maresca; Rebecca Heald

2005-01-01

309

Immune Responses to Foot-and-Mouth Disease DNA Vaccines Can Be Enhanced by Coinjection with the Isatis indigotica Extract  

Microsoft Academic Search

The Isatis indigotica extract is widely used in clinical practice for the treatment of influenza, epidemic hepatitis, epidemic encephalitis B etc. The goal of this study was to investigate whether coinjection of the Isatis indigotica extract with foot-and-mouth disease virus (FMDV) DNA vaccine could increase the protective immune response. Mice were vaccinated twice with either FMDV DNA vaccine plus the

Liang Chen; Tao Lin; HongXin Zhang; YongBo Su

2005-01-01

310

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...

311

Comparison of Eight Methods for the Extraction of Bacillus atrophaeus Spore DNA from Eleven Common Interferents and a Common Swab  

PubMed Central

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

Rose, Helen L.; Dewey, Caroline A.; Ely, Morgan S.; Willoughby, Sarah L.; Parsons, Tanya M.; Cox, Victoria; Spencer, Phillippa M.; Weller, Simon A.

2011-01-01

312

Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis.  

PubMed

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

Linton, Elisabeth; Rahman, Asif; Viamajala, Sridhar; Sims, Ronald C; Miller, Charles D

2012-01-01

313

Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation  

SciTech Connect

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.

Xun Li; Traganos, F.; Melamed, M.R.; Darzynkiewicz, Z. [New York Medical College, Valhalla, NY (United States)

1995-06-01

314

Determination of chloropropanols in foods by one-step extraction and derivatization using pressurized liquid extraction and gas chromatography-mass spectrometry.  

PubMed

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

Racamonde, I; González, P; Lorenzo, R A; Carro, A M

2011-08-10

315

A New Method to Extract Dental Pulp DNA: Application to Universal Detection of Bacteria  

PubMed Central

Background Dental pulp is used for PCR-based detection of DNA derived from host and bacteremic microorganims. Current protocols require odontology expertise for proper recovery of the dental pulp. Dental pulp specimen exposed to laboratory environment yields contaminants detected using universal 16S rDNA-based detection of bacteria. Methodology/Principal Findings We developed a new protocol by encasing decontaminated tooth into sterile resin, extracting DNA into the dental pulp chamber itself and decontaminating PCR reagents by filtration and double restriction enzyme digestion. Application to 16S rDNA-based detection of bacteria in 144 teeth collected in 86 healthy people yielded a unique sequence in only 14 teeth (9.7%) from 12 individuals (14%). Each individual yielded a unique 16S rDNA sequence in 1–2 teeth per individual. Negative controls remained negative. Bacterial identifications were all confirmed by amplification and sequencing of specific rpoB sequence. Conclusions/Significance The new protocol prevented laboratory contamination of the dental pulp. It allowed the detection of bacteria responsible for dental pulp colonization from blood and periodontal tissue. Only 10% such samples contained 16S rDNA. It provides a new tool for the retrospective diagnostic of bacteremia by allowing the universal detection of bacterial DNA in animal and human, contemporary or ancient tooth. It could be further applied to identification of host DNA in forensic medicine and anthropology.

Tran-Hung, Lam; Tran-Thi, Ny; Aboudharam, Gerard; Raoult, Didier; Drancourt, Michel

2007-01-01

316

From soil to leaves--aluminum fractionation by single step extraction procedures in polluted and protected areas.  

PubMed

The paper presents the fractionation of aluminum in the samples of soil and plants of different species using a selective single-step extraction method. The study was conducted in the area located near a chemical plant, which for many years served as a post-crystallization leachate disposal site storing chemical waste (sector I), and in the area around the site: in Wielkopolski National Park, Rogalin Landscape Park and toward the infiltration ponds at the "D?bina" groundwater well-field for the city of Pozna? (Poland) (sector II). The results of aluminum fractionation in samples of soil, leaves and plants showed heavy pollution with aluminum, especially in the water soluble aluminum fraction - Alsw (maximum concentration of aluminum in soil extract was 234.8 ± 4.8 mg kg(-1), in the leaves of Betula pendula it was 107.4 ± 1.8 mg kg(-1) and in the plants of Artemisia vulgaris (root) and Medicago sativa (leaves) it amounted to 464.7 ± 10.7 mg kg(-1)and 146.8 ± 1.2 mg kg(-1) respectively). In addition, the paper presents the problem of organic aluminum fractionation in biological samples and it shows the relationship between aluminum concentration in soil and the analysed woody and herbaceous species. PMID:23651943

Frankowski, Marcin; Zio?a-Frankowska, Anetta; Siepak, Jerzy

2013-05-04

317

General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding.  

PubMed

Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation. PMID:14507688

Lucius, Aaron L; Maluf, Nasib K; Fischer, Christopher J; Lohman, Timothy M

2003-10-01

318

Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli.  

PubMed

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA. PMID:23831957

Haberl, Saša; Jarc, Marko; Strancar, Aleš; Peterka, Matjaž; Hodži?, Duša; Miklav?i?, Damijan

2013-07-06

319

Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using taqman assays  

PubMed Central

SUMMARY Rapid tuberculosis (TB) detection is critical for disease control, and further quantitation of Mycobacterium tuberculosis (Mtb) in sputum is valuable for epidemiological and clinical studies. We evaluated a simple, robust and cost-efficient in-house DNA extraction and downstream taqman approach for detection and quantitation of Mtb genomes from sputum of newly-diagnosed TB patients and non-TB controls. DNA was extracted using guanidine isothiocyanate and silica-based spin columns in less than 2h, stored frozen, and taqman assays were used to detect Mtb with IS6110 and quantify it targeting RD1 and IS1081. The taqmans had a sensitivity > 95% in 108 culture-confirmed TB patients and specificity of 100% in 43 non-TB controls. Genome counts were correlated with the Mycobacterial Growth Indicator Tubes’ (MGIT) time-to-detection values (1/TTD×1000; rho=0.66; p<0.001) in 91 TB patients (33 excluded with MGIT contamination). This linear relationship was nearly identical between mycobacteria isolated from sputum and H37Rv Mtb grown in-vitro to its log phase. TB treatment between 3 and 7 days was associated with lower 1/TTD×1000 values but not with genome counts. Together, our protocol provides rapid, specific, inexpensive and quantitative detection of Mtb DNA in fresh or stored sputa making it a robust tool for prompt TB diagnosis, and with potential use for clinical and epidemiologic studies.

Gomez, Diana I.; Mullin, Caroline S.; Mora-Guzman, Francisco; Crespo-Solis, J. Gonzalo; Fisher-Hoch, Susan P.; McCormick, Joseph B.; Restrepo, Blanca I.

2011-01-01

320

An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing  

PubMed Central

Background Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs. Methodology/Principal Findings We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method. Conclusion Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

Huang, Hui; Gao, Ju; Zhao, You-Jie; Gao, Li-Zhi

2012-01-01

321

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

PubMed Central

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.

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

2011-01-01

322

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

PubMed

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 Fe(3+), 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

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

2011-06-20

323

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

PubMed Central

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.

Razaq, Aamir; Nystrom, Gustav; Str?mme, Maria; Mihranyan, Albert; Nyholm, Leif

2011-01-01

324

High-capacity conductive nanocellulose paper sheets for electrochemically controlled extraction of DNA oligomers.  

PubMed

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 m(2) 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

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

2011-12-15

325

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform  

Microsoft Academic Search

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes\\u000a purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and\\u000a magnetic beads conjugated with CD15\\/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA\\u000a utilizing surface-charge switchable,

Kang-Yi Lien; Chien-Ju Liu; Yi-Chien Lin; Pao-Lin Kuo; Gwo-Bin Lee

2009-01-01

326

Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption  

Microsoft Academic Search

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing

FRANK-MICHAEL C. MULLER; KATHERINE E. WERNER; MIKI KASAI; ANDREA FRANCESCONI; STEPHEN J. CHANOCK; THOMAS J. WALSH

1998-01-01

327

The effect of various stain carriers on the quality and quantity of DNA extracted from dried bloodstains  

Microsoft Academic Search

Bloodstains were made with 200 µl blood on each of 11 different common substrates to examine the effect of the stain carrier on the amount and quality of DNA recoverable. High-molecular-weight DNA was extracted from all samples after 2 days. The yield of DNA from each sample varied considerably, not only between the different stain carriers but also within a

Mechthild Prinz; G. Berghaus

1990-01-01

328

An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.  

PubMed

This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react. PMID:21242033

Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

2010-12-23

329

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

PubMed

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

Demeke, Tigst; Jenkins, G Ronald

2009-09-30

330

CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens  

PubMed Central

Background The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH. Results We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity. Conclusions The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.

2012-01-01

331

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method  

PubMed Central

Background The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. Methods A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. Results Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. Conclusions The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited.

2012-01-01

332

Extraction and quantification of phosphorus derived from DNA and lipids in environmental samples.  

PubMed

Understanding the flux and turnover of phosphorus (P) in the environment is important due to the key role P plays in eutrophication and in the ambition to find cost-effective measures to mitigate it. Orthophosphate diesters, including DNA and phospholipids (PLs), represent a potentially degradable P pool that could support future primary production and eutrophication. In this study, extraction techniques were optimized and combined with colorimetric determination of extracted P to provide a selective quantification method for DNA-P and PL-P in agricultural soil, sediment and composted manure. The proposed method is rapid and reproducible with an RSD of <10%. Recovery, evaluated by spiking the sample matrices with DNA and PL standards, was over 95% for both DNA and PLs. The method can be used for the determination of the pool size of the two organic P fractions. Results show that DNA-P comprises 3.0% by weight of the total P (TP) content in the studied soil, 10.4% in the sediment and 8.4% in the compost samples. The values for PL-P are 0.5%, 6.0% and 1.7% for soil, sediment and compost, respectively. PMID:24054600

Paraskova, Julia V; Rydin, Emil; Sjöberg, Per J R

2013-05-24

333

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA  

PubMed Central

Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/?l) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human ?-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

2011-01-01

334

Comparison of DNA extraction from blood stain and decomposed muscle in STR polymorphism analysis.  

PubMed

Forensic samples that are often degraded and limited in quality cause DNA typing analysis by conventional methods unsuitable. We performed a single tube-multiplex PCR on 9 STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820) and the X-Y homologous gene amelogenin of DNA extracted from six week postmortem blood stain and decomposed muscle by using QIAGEN QIAamp blood or tissue procedure. An automated genetic analyzer based on fluorescent dye technology was used to detect STR allele patterns. The DNA profile of blood stain sample obtained a complete and unambiguous pattern, whereas, that of muscle DNA extracted from QIAamp tissue and Chelex plus QIAamp blood protocols showed detected STR alleles for 70 per cent and 50 per cent of all tested alleles, respectively. The degraded muscle DNA could not yield amplified products of large size STR alleles; CSF1PO, D13S317 and D7S820. However, the analysis which relied upon the PCR-based STR polymorphism analysis and automated genetic analyzer system offers an ideal strategy for forensic identification. PMID:10865412

Rerkamnuaychoke, B; Chantratita, W; Jomsawat, U; Thanakitgosate, J; Pattanasak, N; Rojanasunan, P

2000-03-01

335

Automated microfluidic DNA/RNA extraction with both disposable and reusable components  

NASA Astrophysics Data System (ADS)

An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

2012-01-01

336

The rejoining of double-strand breaks in DNA by human cell extracts.  

PubMed Central

A double-strand DNA break was introduced at a specific site within the lacZ gene of plasmid pUC18 using one of several restriction enzymes, and the plasmid exposed to nuclear extracts from human cell lines. Physical rejoining of DNA was monitored by Southern analysis after gel separation, and the fidelity of rejoining by expression of the lacZ gene after bacterial transformation with the treated plasmid. Breaks at the SalI and EcoRI sites were rejoined by extracts to form circular monomers, but the efficiency of rejoining was much higher at the SalI site. Measurement of rejoining at several adjacent sites having different types of termini, consistently showed a range of efficiencies with 5' 4-base greater than 3' 4-base overhangs and 4-base greater than 2-base greater than no overhang. Similar efficiencies were found for nuclear extracts from transformed cell lines, both from a 'normal' individual and an ataxia-telangiectasia (A-T) patient, and from a non-transformed normal cell culture. In contrast at some sites, especially those with a low rejoin efficiency, the fidelity of rejoining was very much lower for the A-T extracts than for normal cell extracts. Mis-rejoining was, however, unrelated to rejoin efficiency at other sites, suggesting that factors such as the exact sequence at the break site on the molecule may also influence the fidelity of rejoining. Images

North, P; Ganesh, A; Thacker, J

1990-01-01

337

One-step column chromatographic extraction with gradient elution followed by automatic separation of volatiles, flavonoids and polysaccharides from Citrus grandis.  

PubMed

Citrus grandis Tomentosa is widely used in traditional Chinese medicine and health foods. Its functional components include volatiles, flavonoids and polysaccharides which cannot be effectively extracted through traditional methods. A column chromatographic extraction with gradient elution was developed for one-step extraction of all bioactive substances from C. grandis. Dried material was loaded into a column with petroleum ether: ethanol (8:2, PE) and sequentially eluted with 2-fold PE, 3-fold ethanol: water (6:4) and 8-fold water. The elutes was separated into an ether fraction containing volatiles and an ethanol-water fraction containing flavonoids and polysaccharides. The later was separated into flavonoids and polysaccharides by 80% ethanol precipitation of polysaccharides. Through this procedure, volatiles, flavonoids and polysaccharides in C. grandis were simultaneously extracted at 98% extraction rates and simply separated at higher than 95% recovery rates. The method provides a simple and high-efficient extraction and separation of wide range bioactive substances. PMID:24128512

Han, Han-Bing; Li, Hui; Hao, Rui-Lin; Chen, Ya-Fei; Ni, He; Li, Hai-Hang

2013-09-03

338

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

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.

Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

2013-01-01

339

DNA Fingerprinting in a Forensic Teaching Experiment  

ERIC Educational Resources Information Center

|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…

Wagoner, Stacy A.; Carlson, Kimberly A.

2008-01-01

340

Cytotoxicity, apoptosis and DNA damage induced by Alpinia galanga rhizome extract.  

PubMed

Alpinia galanga, or galangal, has been a popular condiment used in Thai and Asian cuisine for many years. However, relatively little is known of the potential beneficial or adverse health effects of this spice. This study was conducted to analyze the capacity of galangal extract to induce cytotoxicity and DNA damage in six different human cell lines including normal and p53-inactive fibroblasts, normal epithelial and tumour mammary cells and a lung adenocarcinoma cell line. We deliberately focused on treatment with the crude aqueous extract of galangal rhizomes, rather than compounds extracted into an organic solvent, to more closely reflect the mode of dietary consumption of galangal. The cell lines displayed a broad range of cytotoxicity. There was no evidence for preferential cytotoxicity of tumour cells, but there was an indication that p53-active cell lines may be more sensitive than their p53-inactive counterparts. The contribution of apoptosis to total cell killing was only appreciable after exposure to 300 microg/mL of extract. Apoptosis appeared to be independent of p53 expression. Exposure to as little as 100 microg/mL galangal extract generated a significant level of DNA single-strand breaks as judged by the single-cell gel electrophoresis technique (comet assay). The three major UV-absorbing compounds in the aqueous extract were identified by mass spectrometry as 1'-acetoxychavicol acetate and its deacetylated derivatives. However, when tested in A549 human lung adenocarcinoma cells, these compounds were not responsible for the cytotoxicity induced by the complete aqueous extract. PMID:17611930

Muangnoi, P; Lu, M; Lee, J; Thepouyporn, A; Mirzayans, R; Le, X C; Weinfeld, M; Changbumrung, S

2007-07-05

341

A spin cartridge system for DNA extraction from paraffin wax embedded tissues.  

PubMed Central

A simple and efficient method of DNA extraction from paraffin wax embedded tissues using a spin cartridge system is described. Such DNAs were shown to be suitable for amplification by the polymerase chain reaction, which targeted two human papillomavirus genes and one globin fragment giving rise to products of 450, 150, and 110 base pairs, respectively. Different human tissues, stored for up to 20 years, were successfully amplified, demonstrating the usefulness of this very simple procedure for retrospective studies.

Pinto, A P; Villa, L L

1998-01-01

342

Detection of tomato yellow leaf curl thailand virus by PCR without DNA extraction  

Microsoft Academic Search

We report the simple and rapid method for detection of tomato yellow leaf curl Thailand virus (TYLCTHV) based on the direct\\u000a capture of virus particles to the surface of a polymerase chain reaction (PCR) tube. This method allowed PCR without the time-consuming\\u000a procedures of DNA extraction from infected plant tissue. A small amount of tomato tissue (?10 mg) was ground

Supaporn Ieamkhang; Lumpueng Riangwong; Orawan Chatchawankanphanich

2005-01-01

343

Dichloromethane as an economic alternative to chloroform in the extraction of DNA from plant tissues  

Microsoft Academic Search

Processing of large numbers smaples of plant tissue samples for molecular mapping and gene tagging requires methods that are\\u000a quick, simple, and cheap, and that eventually can be automated. Organic solvents used for DNA extraction can represent a significant\\u000a proportion of the overall cost. In this study we examined dichloromethane as a replacement for chloroform to be used in combination

Alba Lucía Chaves; Claudia E. Vergara; Jorge E. Mayer

1995-01-01

344

Detection of DNA in virgin olive oils extracted from destoned fruits  

Microsoft Academic Search

Characterization of genetic identity using DNA extracted from olive oil has the potential to facilitate assessment of origin\\u000a and varietal conformity. Such a prospect is particularly interesting in light of the increased regional spread of olive cultivars\\u000a and their various contributions to olive oil mixtures for certification of denomination of origin. Towards this goal, we have\\u000a devised a reliable method

Innocenzo Muzzalupo; Massimiliano Pellegrino; Enzo Perri

2007-01-01

345

Rolling Circle DNA Replication by Extracts of Herpes Simplex Virus Type 1Infected Human Cells  

Microsoft Academic Search

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circlereplicationofcircularduplexDNAmolecules.Theproductsofthereactionarelongerthanmonomerunit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance toDpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on

RAMI SKALITER; ALEXANDER M. MAKHOV; JACK D. GRIFFITH; R. LEHMAN

1996-01-01

346

An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents.  

PubMed

Routine methods of extraction of DNA from blood involve the enrichment of cells by Ficoll-Hypaque gradient centrifugation followed by lysis of the cells with extraction buffer, proteinase K digestion of the lysate, and phenol:chloroform-isoamyl alcohol extraction. These methods generally require large amounts of blood, which poses a problem with pediatric patients. To overcome this, we developed a new method of extracting DNA directly from whole blood. This method involves the treatment of whole blood with an equal volume of NaI (3 M final concentration) followed by chloroform:isoamyl alcohol extraction to clear hemoglobin and cell debris. The clear aqueous layer is then mixed with isopropanol to obtain DNA. A large number of samples can easily be handled by this extraction procedure, as it can be carried out in 30 min and requires only a microcentrifuge. PMID:1955487

Loparev, V N; Cartas, M A; Monken, C E; Velpandi, A; Srinivasan, A

1991-09-01

347

Sarcoptes mite from collection to DNA extraction: the lost realm of the neglected parasite.  

PubMed

Sarcoptes mite from collection to DNA extraction forms the cornerstone for studies on Sarcoptes scabiei. Whilst the new science era took a shy leap into the different facets of mite studies, the cornerstone was almost entirely neglected. Mite collection, cleaning, storage and DNA extraction were, basically, humble attempts to extrapolate, adapt, modify or 'pirate' those existing methods to the peculiarities of Sarcoptes research. These aspects usually constituted few lines, bashfully mentioned, in the materials and methods section of some papers, which arose in unique problems concerning cost-effectiveness, time profitability, safety and even worse, the credibility of the results, creating contradictory conclusions in some cases. This 'noisy' situation encouraged us to collect, classify and review, for the first time to our knowledge, some aspects relating to studies on Sarcoptes mite from collection to DNA extraction, which will be useful for further studies on Sarcoptes, and have implications for the effective control of the diseases Sarcoptes mite causes. Further studies are needed, especially to compare the profitability, safety, sensibility and specificity of the different methods of this neglected realm of the ubiquitous ectoparasite. PMID:19159955

Alasaad, S; Rossi, L; Soriguer, R C; Rambozzi, L; Soglia, D; Pérez, J M; Zhu, X Q

2009-01-22

348

Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina.  

PubMed

Eight crude extracts from seven Argentine plants with cancer-related ethnobotanical uses have been subjected to a bioscreening study to detect cytotoxic activity. The plants studied were: Aristolochia triangularis, Baccharis grisebachii, Bolax gummifera, Eupatorium hecatanthum, Erythrina crista-galli, Pterocaulon polystachium and Salpichroa origanifolia. Crown gall tumour inhibition, DNA interaction and cytotoxicity towards KB cells were assayed using the potato disc, the DNA-methyl green (DNA-MG) and the KB cells cytotoxicity bioassays respectively. The results obtained indicate that A. triangularis (ED50=47 microg/ml), B. gummifera (ED50=32 microg/ml) and E. hecatanthum (ED50=35 microg/ml) contained cytotoxic compounds against KB cells. All of the plants studied inhibited the growth of crown gall tumours, showing correlation between the experimental data and the uses reported for these plants. Moreover, the results obtained for the extracts of E. hecatanthum and P. polystachium indicate the presence of compounds that interact with DNA (48 and 22% of absorbance decrease, respectively). The results obtained suggest that cytotoxicity could play an important role in the activities claimed for the plants under study. PMID:10904157

Mongelli, E; Pampuro, S; Coussio, J; Salomon, H; Ciccia, G

2000-07-01

349

Aqueous extracts of Lippia turbinata and Aloysia citriodora (Verbenaceae): assessment of antioxidant capacity and DNA damage.  

PubMed

The aim of the present work was to make a contribution to the knowledge of aqueous extracts of Lippia turbinata and Aloysia citriodora (Verbenaceae; infusion and decoction) in relation with the establishment of its antioxidant activity and lack of DNA damage, for its potential use in therapeutics. The cytogenotoxic profile was evaluated through genotoxic biomarkers such as mitotic index, cellular proliferation kinetics, sister chromatid exchanges, single-cell gel electrophoresis assay, and micronucleus test in human peripheral blood lymphocyte cultures. No statistical differences were found (P > .05) between control and exposed cultures, even between both aqueous extracts. The total antioxidant capacity was shown to be higher in the decoction than in the infusion and both aqueous extracts protected against protein carbonylation and lipid peroxidation, the decoction being more efficient than the infusion (P < .005). These results suggest the safe use of these medicinal plants as chemoecologic agents in therapeutics. PMID:22427199

Portmann, Erika; Nigro, Marcela M López; Reides, Claudia G; Llesuy, Susana; Ricco, Rafael A; Wagner, Marcelo L; Gurni, Alberto A; Carballo, Marta A

2012-03-16

350

Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens  

PubMed Central

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Liu, Christina; Lin, Juan; Ye, Kenny; Kim, Ryung; Hazan, Rachel; Rohan, Thomas; Fineberg, Susan; Loudig, Olivier

2012-01-01

351

Quantitative PCR analysis of blood- and saliva-specific microRNA markers following solid-phase DNA extraction.  

PubMed

The use of mRNA for the identification of body fluids is of particular interest in forensic science, and increasing support has been demonstrated for the use of microRNA (miRNA) analysis. MiRNA is more stable than mRNA and has been shown to be differentially expressed in body fluids. No studies involving miRNA analysis from previously extracted DNA samples have yet been reported. The aim of this experiment was to determine if it was possible to conduct miRNA analysis on samples that were previously extracted using standard DNA extraction. Blood and saliva samples were extracted using DNA and RNA kits, followed by cDNA synthesis, and then underwent quantitative PCR analysis. A direct comparison of ?Ct values shows a larger abundance of miRNA following DNA extraction as opposed to total RNA extraction for both blood- and saliva-specific markers. By carrying out a comparison between the amounts of said markers, it could be seen that the expression of the blood-specific marker was higher in blood than in saliva, and vice versa for the saliva-specific marker. The results obtained could have a profound impact on cases for which the sample has already undergone DNA extraction, such as in cold cases. PMID:23333269

Omelia, Emma J; Uchimoto, Mari L; Williams, Graham

2013-01-16

352

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces.  

PubMed

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers. PMID:18162191

Nechvatal, Jordan M; Ram, Jeffrey L; Basson, Marc D; Namprachan, Phanramphoei; Niec, Stephanie R; Badsha, Kawsar Z; Matherly, Larry H; Majumdar, Adhip P N; Kato, Ikuko

2007-11-21

353

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.  

PubMed

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers. PMID:16302208

Nasiri, H; Forouzandeh, M; Rasaee, M J; Rahbarizadeh, F

2005-01-01

354

A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping  

PubMed Central

Background Puccinia striiformis f.sp. tritici (PST), an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing. Findings We report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy. Conclusion These two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.

2011-01-01

355

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...

356

Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts  

PubMed Central

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) has a well-established role in epigenetic regulation through the recognition of various histone marks and interaction with chromatin-modifying proteins. However, its function in regulating cell cycle progression remains poorly understood and has been largely attributed to a role in transcriptional regulation. In this study we have used Xenopus laevis egg extracts to analyse Uhrf1 function in DNA replication in the absence of transcriptional influences. We demonstrate that removal of Uhrf1 inhibits chromosomal replication in this system. We further show that this requirement for Uhrf1, or an associated factor, occurs at an early stage of DNA replication and that the consequences of Uhrf1 depletion are not solely due to its role in loading Dnmt1 onto newly replicated DNA. We describe the pattern of Uhrf1 chromatin association before the initiation of DNA replication and show that this reflects functional requirements both before and after origin licensing. Our data demonstrate that the removal of Xenopus Uhrf1 influences the chromatin association of key replication proteins and reveal Uhrf1 as an important new factor required for metazoan DNA replication.

Taylor, Elaine M.; Bonsu, Nicola M.; Price, R. Jordan; Lindsay, Howard D.

2013-01-01

357

Endonuclease activities in extracts of Micrococcus luteus that act on gemma-irradiated DNA.  

PubMed

Several protein fractions containing endonuclease activity against gemma-irradiated DNA (gamma-endonuclease) were isolated from M. luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxyapatite. Five peaks of gamma-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behavior of the fractions. There was no detectable activity of U.V.-endonuclease in the fractions with gamma-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The gamma-endonuclease is stimulated by, but is not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the gamma-endonuclease carry 3'OH and 5' phosphate end groups. PMID:300726

Schön-Bopp, A; Schäfer, G; Hagen, U

1977-03-01

358

Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR?  

PubMed Central

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.

Munoz-Cadavid, C.; Rudd, S.; Zaki, S. R.; Patel, M.; Moser, S. A.; Brandt, M. E.; Gomez, B. L.

2010-01-01

359

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods.  

PubMed

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 with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended. PMID:16359805

Gonzales, J L; Loza, A; Chacon, E

2005-12-15

360

Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products.  

PubMed

Areca nut is a carcinogen to humans and has been strongly associated with oral premalignant and malignant diseases. Previous studies speculated the presence of unknown direct-acting mutagens present in aqueous extracts of areca nut. We hypothesized whether any direct-acting alkylating agents are present in areca nut and its commercial products. In this study, calf thymus DNA was treated with four different aqueous extracts obtained from unripe and ripe areca nuts or their commercial products, namely, pan masala (without tobacco) and gutkha (with tobacco). Three N-alkylated purines including N7-methylguanine (N7-MeG), N3-methyladenine (N3-MeA), and N7-ethylguanine (N7-EtG) were detected using sensitive and specific isotope-dilution liquid chromatography-tandem-mass spectrometry (LC-MS/MS) methods. The results showed that four types of aqueous extracts significantly induced the formation of N7-MeG and N3-MeA in a linear dose-response manner. Extracts from unripe areca nut exhibited higher methylating potency than those of ripe areca nut, while gutkha had higher methylating potency than pan masala. Meanwhile, gutkha made with areca nut and tobacco, was the only extract found to induce the formation of N7-EtG. Overall, this study first demonstrated that the presence of direct-acting alkylating agents in areca nut and its commercial products exist at a level that is able to cause significant DNA damage. Our findings may provide another mechanistic rationale for areca nut-mediated oral carcinogenesis and also highlight the importance and necessity of the identification of these direct-acting alkylating agents. PMID:23033867

Hu, Chiung-Wen; Chao, Mu-Rong

2012-10-16

361

Termination Point of Replication of Colicin E1 Plasmid DNA in Cell Extracts*  

PubMed Central

Closed-circular monomeric molecules were one of the major products of replication of colicin E1 plasmid DNA in cell extracts. However, when the plasmid DNA synthesized in the reaction mixture was labeled for 3 min after 27 min of incubation, most of the label was found in open-circular molecules. The open-circular molecules were converted to closed-circular molecules upon further incubation. The newly replicated open-circular molecules had a nick or small gap in their newly synthesized strand. The interruption was located at approximately 20% of the molecular length from the single site of cleavage by restriction endonuclease EcoR1 and at or very close to the origin of replication. The addition of 10 mM nicotinamide mononucleotide instead of nicotinamide adenine dinucleotide to extracts did not significantly affect the kinetics of the colicin E1 plasmid DNA synthesis. However, in the presence of nicotinamide mononucleotide the formation of completely replicated closed-circular molecules was suppressed and, instead, open-circular molecules accumulated with an interruption in the newly synthesized strand at the termination point of replication, which was located at or very close to the origin of replication.

Sakakibara, Yoshimasa; Tomizawa, Jun-Ichi

1974-01-01

362

Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method  

Microsoft Academic Search

Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time

A. M. NSUBUGA; M. M. ROBBINS; A. D. ROEDER; P. A. MORIN; C. BOESCH; L. VIGILANT

2004-01-01

363

Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis  

Microsoft Academic Search

The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in

Lotta Nylund; Hans G. H. J. Heilig; Seppo Salminen; Willem M. de Vos; Reetta Satokari

2010-01-01

364

Procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder  

Microsoft Academic Search

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction,

M. A. Marko; R. Chipperfield; H. C. Birnboim

1982-01-01

365

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

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

Jan, Ji?í; Borovec, Jakub; Kopá?ek, Ji?í; Hejzlar, Josef

2012-11-21

366

A SYBR green-based real-time polymerase chain reaction protocol and novel DNA extraction technique to detect Xylella fastidiosa in Homalodisca coagulata.  

PubMed

Homalodisca coagulata Say (Hemiptera: Cicadellidae) is a major agronomic pest because it transmits Xylella fastidiosa (Wells), the bacterium that causes Pierce's disease of grapevine. The ability to easily detect X. fastidiosa in populations of H. coagulata facilitates epidemiological studies and development of a monitoring program supporting disease management. Such a program depends on a detection protocol that is rapid, reproducible, and amenable to large sample sizes, while remaining sensitive enough to detect low amounts of pathogen DNA. In this study, we developed an improved method to speed DNA extraction by implementing a simple vacuum step that replaces labor- and time-intensive maceration of tissue samples and that is compatible with manufactured DNA extraction kits. Additionally, we have developed a SYBR Green-based real-time (RT)-polymerase chain reaction (PCR) system, which uses traditional PCR primers that are relatively inexpensive and effective. Using this extraction/RT-PCR system, we found no statistically significant differences in the detection of X. fastidiosa among samples that were either immediately extracted or stored dry or in mineral oil for 10 d at -4 degrees C. In further testing, we found no significant reduction in detection capabilities for X. fastidiosa-fed H. coagulata left in the sun on yellow sticky cards for up to 6 d. Therefore, we recommend a field-based detection system that includes recovery of H. coagulata from sticky traps for up to 6 d after trapping, subsequent freezing of samples for as long as 10 d before vacuum extraction is performed, and detection of the bacterium by SYBR Green-based RT-PCR. PMID:16022291

Bextine, Blake; Blua, Matthew; Harshman, Dave; Miller, Thomas A

2005-06-01

367

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil  

Microsoft Academic Search

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)

K. Hjort; M. Bergstrom; M. F. Adesina; J. K. Jansson; K. Smalla; S. Sjoling

2009-01-01

368

A fast one-step extraction and UPLC-MS/MS analysis for E2/D 2 series prostaglandins and isoprostanes.  

PubMed

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 the 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

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

2013-02-12

369

Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.  

PubMed

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 H2O2-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 Na2EDTA/g). PMID:23871827

Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

2013-07-16

370

Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo  

PubMed Central

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.

Ke, Yuebin; Xu, Xinyun; Wu, Shuang; Huang, Juan; Misra, Hara; Li, Yunbo

2013-01-01

371

Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album's ethanolic leaf extract.  

PubMed

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

Elif Korcan, S; Aksoy, Onur; Erdo?mu?, S Feyza; Ci?erci, ? Hakk?; Konuk, Muhsin

2012-08-13

372

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

PubMed Central

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 MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

1992-01-01

373

DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer.  

PubMed

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow enhanced extraction of DNA from fluid suspensions. Characterization of the synthesis revealed linear doubling of the surface amine charge from generations one through five starting with an amino silane initiator. Furthermore, transmission electron microscopy revealed clear dispersion of the single domain magnetite in aqueous solution. The dendrimer modified magnetic particles have been used to carry out magnetic separation of DNA. Binding and release efficiencies increased with the number of generations and those of bacterial magnetite modified with six generation dendrimer were 7 and 11 times respectively as many as those of bacterial magnetite modified with only amino silane. PMID:12615391

Yoza, Brandon; Arakaki, Atsushi; Matsunaga, Tadashi

2003-03-20

374

Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition  

PubMed Central

Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g?1 using Griffiths and 4.25×106 cells g?1 using FastDNA® Spin kit.

Pontiroli, Alessandra; Travis, Emma Rachel; Sweeney, Francis Patrick; Porter, David; Gaze, William Hugo; Mason, Sam; Hibberd, Victoria; Holden, Jennifer; Courtenay, Orin; Wellington, Elizabeth Margaret Helen

2011-01-01

375

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis).  

PubMed

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as "fecal hairs". In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I-the highest-quality DNA, grade II--high-quality DNA, and grade III--poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. PMID:19358318

Zhang, Wenping; Zhang, Zhihe; Xu, Xiao; Wei, Kun; Wang, Xiaofang; Liang, Xu; Zhang, Liang; Shen, Fujun; Hou, Rong; Yue, Bisong

376

Diminution of free radical induced DNA damage by extracts/fractions from bark of Schleichera oleosa (Lour.) Oken.  

PubMed

The present study was undertaken to investigate the effect of extracts of Schleichera oleosa (Sapindaceae) for its cytotoxic and hydroxyl radical-scavenging activities. The bark of the tree was used to prepare extracts with different solvents (i.e., hexane, chloroform, ethyl acetate, methanol, and water). The extracts were initially assessed for their in vitro cytotoxicity potential in the sulforhodamine B dye assay against cell lines, such as 502713 (colon), SW-620 (colon), HCT-15 (colon), A-549 (lung), HEP-2 (liver), SK-NS-H (central nervous system), and IMR-32 (neuroblastoma). It was observed that the water extract was effective against all the three colon cancer cell lines, while only methanol and water extracts were effective against A-549 (lung) and HEP-2 (liver), respectively. As DNA damage is one of the hallmarks of cell death, so the extracts were assessed for their ability to scavenge hydroxyl radicals, in the deoxyribose degradation assay (site- and nonsite specific) as well as their protective effect against the hydroxyl radical-induced DNA damage in the plasmid nicking assay, using pBR322. It was observed that all the extracts, except chloroform and hexane, exhibited relatively greater antioxidant activity in the nonsite-specific than in the site-specific assay. Similarly, the extracts were also found to be effective in inhibiting the hydroxyl radical-induced unwinding of supercoiled DNA, which further confirmed the hydroxyl radical-scavenging ability of the extracts in the deoxyribose degradation method. PMID:20545578

Thind, Tarunpreet S; Rampal, Geetanjali; Agrawal, Satyam K; Saxena, Ajit K; Arora, Saroj

2010-10-01

377

Multiple components are involved in the efficient joining of double stranded DNA breaks in human cell extracts.  

PubMed Central

We describe a rapid and efficient in vitro system for the rejoining of double stranded breaks in DNA based on extracts of human 293 cells. Using this system as an assay, we have separated the nuclear extract into several components involved in break rejoining. The unfractionated system can convert approx. 100% of the input DNA, linearized with a restriction enzyme, to high molecular weight material at low temperature (17 degrees C), and at the physiological temperature of 37 degrees C we have shown that competing activities in the extract can also act on the DNA template. We present the fractionation of the extract and the partial purification of a novel factor which will stimulate a crude rejoin activity and in addition increases the activity of purified DNA ligase I. We have also partially purified the break joining activity and show that the chromatographic properties do not directly correspond with the three DNA ligases previously described, indicating that the activity observed may not be due to a single enzyme species. By studying the rejoining of double stranded DNA breaks as a biochemical process, we have demonstrated that the efficient joining of such breaks requires factors in addition to DNA ligases. Images

Fairman, M P; Johnson, A P; Thacker, J

1992-01-01

378

Evaluation of DNA Extraction Techniques for Detecting Mycobacterium tuberculosis Complex Organisms in Asian Elephant Trunk Wash Samples?  

PubMed Central

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

Kay, Meagan K.; Linke, Lyndsey; Triantis, Joni; Salman, M. D.; Larsen, R. Scott

2011-01-01

379

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae.  

PubMed

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 approximately 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

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-02-06

380

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae  

PubMed Central

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.

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-01-01

381

Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.  

PubMed Central

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.

Thomas, D C; Svoboda, D L; Vos, J M; Kunkel, T A

1996-01-01

382

Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.  

PubMed

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 (C T ) 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 5.5×10(4) 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

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

2013-02-27

383

Transcription of host-substituted simian virus 40 DNA in whole cells and extracts.  

PubMed Central

Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40.

Kuff, E L; Ferdinand, F J; Khoury, G

1978-01-01

384

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

PubMed

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 Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples. PMID:22182593

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

2011-12-17

385

Ddc1 checkpoint protein and DNA polymerase ? interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae  

Microsoft Academic Search

To characterize proteins that interact with base excision\\/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2?-deoxycytidine-5?-triphosphate, and [32P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at

Maria V. Sukhanova; Claudine D’Herin; Patricia Auffret van der Kemp; Vladimir V. Koval; Serge Boiteux; Olga I. Lavrik

2011-01-01

386

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability  

PubMed Central

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 drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Methods DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. Results The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. Conclusions The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

2013-01-01

387

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants.  

PubMed Central

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic. Images

Rowe, D T; Graham, F L

1983-01-01

388

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants.  

PubMed

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic. PMID:6854738

Rowe, D T; Graham, F L

1983-06-01

389

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil  

SciTech Connect

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.

Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

2009-09-01

390

Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations  

PubMed Central

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.

2012-01-01

391

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.  

PubMed Central

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

Andre, J; Raynaud, A; Rochefort, H

1980-01-01

392

Detection of tomato yellow leaf curl Thailand virus by PCR without DNA extraction.  

PubMed

We report the simple and rapid method for detection of tomato yellow leaf curl Thailand virus (TYLCTHV) based on the direct capture of virus particles to the surface of a polymerase chain reaction (PCR) tube. This method allowed PCR without the time-consuming procedures of DNA extraction from infected plant tissue. A small amount of tomato tissue (approximately 10 mg) was ground in extraction buffer to release viruses from plant tissues. The constituents of the plant extract that might inhibit PCR activity were discarded by washing the tube with PBST buffer before adding the PCR mixture to the tube. This method was used for detection of TYLCTHV with plant sap solution diluted up to 1:20,000 and was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method. In addition, this method can be used for detection of TYLCTHV in viruliferous whiteflies. The PCR tubes with captured TYLCTHV could be used for PCR, after storage at 4 degrees C for 4 wk. The method presented here was used for detection of begomoviruses in cucurbit and pepper. In addition, this method was effectively used to detect papaya ringspot virus in papaya and zucchini yellow mosaic virus in cucumber by reverse transcriptase (RT)-PCR. PMID:16230773

Ieamkhang, Supaporn; Riangwong, Lumpueng; Chatchawankanphanich, Orawan

2005-11-01

393

Extracting DNA from the gut microbes of the termite (Zootermopsis nevadensis).  

PubMed

Termites are among the few animals known to have the capacity to subsist solely by consuming wood. The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson & Breznak, 1983). Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host. The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi & Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR). PMID:18979000

Matson, Eric; Ottesen, Elizabeth; Leadbetter, Jared

2007-05-28

394

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis nevadensis)  

PubMed Central

Termites are among the few animals known to have the capacity to subsist solely by consuming wood. The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson & Breznak, 1983). Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host. The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi & Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Matson, Eric; Ottesen, Elizabeth; Leadbetter, Jared

2007-01-01

395

A validation study for the extraction and analysis of DNA from human nail material and its application to forensic casework.  

PubMed

A validation study was conducted to demonstrate that deoxyribonucleic acid (DNA) could be successfully extracted from human nail material and analyzed using short tandem repeat (STR) profiling and/or mitochondrial DNA (mtDNA) sequencing. This study involved the development of a DNA extraction protocol that includes a cleaning procedure designed to remove external contaminants (e.g., biological, chemical). This protocol was used to test human nail material that had been soaked in whole blood from a second donor and coated with gold-palladium to simulate scanning electron microscopic analysis. The results showed no indication of a mixture and were consistent with that of the nail donor. Fresh human nail material usually yielded both STR profiles and mtDNA sequence information; however, aged human nail material (approximately eight years old) yielded only mtDNA sequence information. Upon completion of the validation study, the extraction protocol was used for the analysis of a torn fingernail fragment recovered from the scene of a violent homicide in 1983. A partial STR profile and mtDNA sequence information indicated that the fingernail fragment was excluded as originating from the suspect and was, in fact, consistent with originating from one of the victims. PMID:10486958

Anderson, T D; Ross, J P; Roby, R K; Lee, D A; Holland, M M

1999-09-01

396

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 Central

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.

Brandfass, Christoph; Karlovsky, Petr

2008-01-01

397

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

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 debr