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1

DNA Extraction  

NSDL National Science Digital Library

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

Stephens, Janice; Leach, Jan

2011-01-01

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

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

Microsoft Academic Search

SUMMARY 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 using previously published oligonucleotide primers specific to X. fastidiosa. The procedure does not require the use of phenol,

Q. Huang; J. Bentz; J. L. Sherald

2006-01-01

4

A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds.  

PubMed

Extraction of high-quality RNA from Arabidopsis seeds has been a challenge. Here we report a two-step TRIzol-based procedure for RNA extraction from Arabidopsis siliques and dry seeds. This procedure employs a modified, high pH (pH 9.5) extraction buffer. High pH plus the addition of either DTT or beta-mercaptoethanol in the extraction buffer effectively inhibits RNase activity during the extraction, and removes most polysaccharides, polyphenols and other insoluble material. TRIzol reagent was subsequently used to purify the RNA. Using this procedure we isolated high-quality DNA-free RNA samples without DNase I treatment from Arabidopsis seeds or siliques in less than 3 h. PMID:20108272

Meng, Ling; Feldman, Lewis

2010-02-01

5

DNA and DNA Extraction  

NSDL National Science Digital Library

The DNA structure and how it makes up genes and chromosomes in the cell. This is the second of a series of seven animations that detail the process of crop genetic engineering. To begin at the beginning, see Overview of Crop Genetic Engineering. (To go to the next animation, go to Gene Cloning.)

6

Onion DNA Extraction  

NSDL National Science Digital Library

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

Hays, Lana

2009-01-01

7

Wheat Germ DNA Extraction  

NSDL National Science Digital Library

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

Hays, Lana

2009-01-01

8

DNA Extraction Virtual Lab  

NSDL National Science Digital Library

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

2006-01-01

9

Thymus DNA Extractions  

NSDL National Science Digital Library

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

Hays, Lana

2009-01-01

10

A Quick and Safe Method for Fungal DNA Extraction  

Microsoft Academic Search

A number of methods have been developed for genomic DNA extraction from fungal tissues. In general, DNA extraction methods consist of several steps, such as preparation of starting materials, generation of cell lysates, elimination of contaminants, and collection of DNA. Conventional fungal DNA extraction methods that are derived from plant genomic DNA extraction methods (Rogers and Bendich, 1985) require fresh

Myoung-Hwan Chi; Sook-Young Park; Yong-Hwan Lee

2009-01-01

11

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

12

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

13

Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment.  

PubMed

Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases. PMID:21855956

Ahmad, Arine Fadzlun; Lonnen, James; Andrew, Peter W; Kilvington, Simon

2011-10-15

14

Introduction to DNA Extractions  

NSDL National Science Digital Library

This lab exercise, authored by Lana Hays of Access Excellence at the National Health Museum, giving instructions for the extraction of DNA from several different starting materials. The lab employs everyday material which can be found at your local grocery store. The exercise is designed for the 6-12 grade level.

Hays, Lana

2009-11-04

15

A Simply Fruity DNA Extraction  

NSDL National Science Digital Library

In this activity, learners extract DNA from a strawberry and discover that DNA is in the food they eat. Learners use simple materials to break open the cells of a strawberry and see DNA with their very own eyes.

Workshop, Mission S.

2012-01-01

16

Ancient DNA extraction from plants.  

PubMed

A variety of protocols for DNA extraction from archaeological and paleobotanical plant specimens have been proposed. This is not surprising given the range of taxa and tissue types that may be preserved and the variety of conditions in which that preservation may take place. Commercially available DNA extraction kits can be used to recover ancient plant DNA, but modifications to standard approaches are often necessary to improve yield. In this chapter, I describe two protocols for extracting DNA from small amounts of ancient plant tissue. The CTAB protocol, which I recommend for use with single seeds, utilizes an incubation period in extraction buffer and subsequent chloroform extraction followed by DNA purification and suspension. The PTB protocol, which I recommend for use with gourd rind and similar tissues, utilizes an overnight incubation of pulverized tissue in extraction buffer, removal of the tissue by centrifugation, and DNA extraction from the buffer using commercial plant DNA extraction kits. PMID:22237524

Kistler, Logan

2012-01-01

17

Extraction of DNA from paleofeces.  

PubMed

Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplified version of a protocol to extract DNA from paleofecal remains. PMID:22237519

Kuch, Melanie; Poinar, Hendrik

2012-01-01

18

Exploring DNA Extraction Erica Butts  

E-print Network

quantity) Known quant value: 52.44 ng/�L Ranges from 1500 ng to 100 ng Human epithelial cell lines*: 100 L of a cell suspension swabbed from Teflon tube (n=12 per quantity) Number of cells determined through flow to precipitate DNA � Rehydrated with 100 �L TE #12;Applied Genetics Blood Extracted DNA Cells Extraction

Perkins, Richard A.

19

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. PMID:22166595

2011-01-01

20

Multifractal analysis and feature extraction of DNA sequences  

Microsoft Academic Search

This paper presents feature extraction and estimations of multifractal measures for deoxyribonucleic acid (DNA) sequences, and demonstrates the intriguing possibility of identifying biological functionality using information contained within the DNA sequence. We have developed a technique that seeks patterns or correlations in the DNA sequence at a higher level. The technique has three main steps: (i) transforms the DNA sequence

Witold Kinsner; Hong Zhang

2009-01-01

21

DNA Extraction & Staging Laboratory (DESL)  

Cancer.gov

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

22

Enhanced sampling simulations of DNA step parameters.  

PubMed

A novel approach for the selection of step parameters as reaction coordinates in enhanced sampling simulations of DNA is presented. The method uses three atoms per base and does not require coordinate overlays or idealized base pairs. This allowed for a highly efficient implementation of the calculation of all step parameters and their Cartesian derivatives in molecular dynamics simulations. Good correlation between the calculated and actual twist, roll, tilt, shift, and slide parameters is obtained, while the correlation with rise is modest. The method is illustrated by its application to the methylated and unmethylated 5'-CATGTGACGTCACATG-3' double stranded DNA sequence. One-dimensional umbrella simulations indicate that the flexibility of the central CG step is only marginally affected by methylation. 2014 Wiley Periodicals, Inc. PMID:25303338

Karolak, Aleksandra; van der Vaart, Arjan

2014-12-15

23

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.

Marek, Edmund; Mulville, Charlotte; Bell, Don

2009-01-01

24

How to Extract DNA From Anything Living  

NSDL National Science Digital Library

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

Utah, University O.

2008-01-01

25

DNA extraction from herbarium specimens.  

PubMed

With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP). PMID:24415470

Drbkov, Lenka Zvesk

2014-01-01

26

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. PMID:20039786

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

2009-01-01

27

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

28

A developed DNA extraction method for different soil samples.  

PubMed

Four DNA extraction methods namely SDS-hyperhaline method (I), modified SDS-hyperhaline method (II), indirect method (III), alkaline lysis method (IV) were evaluated by comparing DNA yield, spectrophotometric quality, genomic integrity and PCR suitability in this paper. The results showed that high DNA yields were obtained by method I, II and IV. However, higher quality of DNA was gained by method III and IV. Based on the results of the Pulsed-Field Gel Electrophoresis (PFGE), the completeness of DNA extracted by method IV was the best. About 6.0 microg DNA can be recovered from 1.0 g soil by method IV which involved to lysis cell by SDS and to precipitate impurities by adding potassium acetate and magnesium chloride Therefore, it is confirmed that method IV is a novel, reliable and versatile method for large-scale DNA extraction involving less purification steps for various soil samples. PMID:20586066

Hu, Yingchang; Liu, Zhiheng; Yan, Jianfang; Qi, Xiaohui; Li, Jing; Zhong, Shiqi; Yu, Jicheng; Liu, Qiu

2010-08-01

29

A rapid and simple method for extracting yeast mitochondrial DNA  

Microsoft Academic Search

A rapid method for the extraction of yeast mitochondrial DNA (mtDNA) is described. In comparison with previous methods, it simplifies several steps, does not require either the isolation of mitochondria or phenol treatment and is less time consuming. This protocol gives a high yield of pure mtDNA (50120 g from a 100-ml culture), which can be directly used in various

Ali Gargouri; M. Curie

1989-01-01

30

DNA Extraction and Quantitation for Forensic Analysts  

NSDL National Science Digital Library

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

2011-05-18

31

Nondestructive DNA extraction from museum specimens.  

PubMed

Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA extraction from other types of specimens such as arthropods. PMID:22237527

Hofreiter, Michael

2012-01-01

32

[DNA extraction from hard dental tissues].  

PubMed

Two different standard ways of DNA extraction (salting out and phenol-chloroform methods) were assayed in order to recovery nucleic acids from dental tissues. The DNA extracted was tested for purity by means of transverse alternating field electrophoresis (TAFE) using Saccharomyces cerevisiae chromosomes as markers. Both extraction methods give similar qualitative and quantitative results being a DNA yield from hard dental tissues approximately 30% of those extracted from the whole tooth. Our results indicate salting out as a preferable method due to its rapidity and usefulness. PMID:8510614

Avitabile, M; Dell'Osso, G; Ras, R; Tripi, F; Campagna, N E; Magr, G A; Sciacca, G; Ras, A

1993-01-01

33

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

34

A simplified universal genomic DNA extraction protocol suitable for PCR.  

PubMed

Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 ?g/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies. PMID:21476197

Wang, T Y; Wang, L; Zhang, J H; Dong, W H

2011-01-01

35

DNA extraction from rice endosperm (including a protocol for extraction of DNA from ancient seed samples).  

PubMed

Deoxyribonucleic acid (DNA) extracted from endosperm can be effectively used for rapid genotyping using seed tissue, to evaluate seed quality from packaged grains and to determine the purity of milled grains. Methods outlined here are optimal procedures to isolate DNA from endosperm tissue of modern rice grains and of aged rice remains preserved between 50 and 100 years. The extracted DNA can be used to amplify regions of chloroplast genomic DNA (ctDNA), mitochondrial genomic DNA (mtDNA), and nuclear genomic DNA using standard PCR protocols. In addition, we describe an optimal procedure to process archaeological grain specimens, aged for a couple of thousand years, to isolate DNA from these ancient samples, referred to here as ancient DNA (aDNA). The aDNA can be successfully amplified by PCR using appropriate primer pairs designed specifically for aDNA amplification. PMID:24243191

Mutou, Chiaki; Tanaka, Katsunori; Ishikawa, Ryuji

2014-01-01

36

DNA extraction techniques for use in education.  

PubMed

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 readily available source of cells for DNA extraction and can be harvested in a painless, noninvasive manner. Seven criteria were established to evaluate the protocol: Safety, DNA yield, DNA quality/stability, cost, user friendliness, reliability, and time. To identify the optimum conditions for each stage of the protocol (cell harvest, lysis, purification, and precipitation), each was investigated separately, and an adaptation of the fast-boiling protocol was used for the remaining stages. A validation study was undertaken with the optimized protocol to assess its performance when conducted by a group of students in a classroom setting. The optimum protocol used an isotonic Lucozade Hydro Active Fitness Water (HAFW) mouthwash. Lysis was achieved using a TE (10 mM Tris-HCl, 1 mM EDTA, pH 8) + 1% Sodium Dodecyl Sulphate (SDS) buffer. Protein was then digested using Proteinase K (Qiagen Inc., UK) at 56C for 10 min. The DNA was then precipitated with sodium chloride and absolute ethanol. This protocol achieved an increase in DNA yield using readily available equipment and reagents at a lower per capita cost and is simple to use. PMID:21567818

Hearn, R P; Arblaster, K E

2010-05-01

37

Crowdsourcing Step-by-Step Information Extraction to Enhance Existing How-to Videos  

E-print Network

- ing, and text/visual analysis techniques. Extracted step information can be used to help learners Harvard SEAS Cambridge, MA USA kgajos@eecs.harvard.edu ABSTRACT Millions of learners today use how-by-step annotations. We first performed a formative study to verify that annota- tions are actually useful to learners

38

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 PMID:10883670

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

2000-01-01

39

Fern spore extracts can damage DNA.  

PubMed

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

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

2000-07-01

40

An Efficient Method for Genomic DNA Extraction from Different Molluscs Species  

PubMed Central

The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others. PMID:22174651

Pereira, Jorge C.; Chaves, Raquel; Bastos, Estela; Leitao, Alexandra; Guedes-Pinto, Henrique

2011-01-01

41

A Simple Method to Extract DNA from Hair Shafts Using Enzymatic Laundry Powder  

PubMed Central

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

Liu, Jinchuan; Jiang, Xiaoling; Li, Sicong; Yang, Shuming; Chen, Ailiang

2013-01-01

42

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

43

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

PubMed Central

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

2014-01-01

44

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads.  

PubMed

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract. PMID:17269682

Oktem, Huseyin Avni; Bayramoglu, Gulay; Ozalp, V Cengiz; Arica, M Yakup

2007-01-01

45

Crowdsourcing Step-by-Step Information Extraction to Enhance Existing How-to Videos  

E-print Network

, and text/visual analysis techniques. Extracted step information can be used to help learners navigate how}@mit.edu pg@cs.rochester.edu kgajos@eecs.harvard.edu ABSTRACT Millions of learners today use how-to videos. We first performed a formative study to verify that annota tions are actually useful to learners. We

Chen, Yiling

46

[Research advances on DNA extraction methods from peripheral blood mononuclear cells].  

PubMed

DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized. PMID:25338615

Wang, Xiao-Ying; Yu, Chen-Xi

2014-09-01

47

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

2010-03-01

48

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

SciTech Connect

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

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

2009-07-31

49

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues  

PubMed Central

Background Nucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA. Principal Findings Regarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was <3g in a final volume of 25l and the quality was high (260/280nm ratio = 1.890.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M.tuberculosis, M.avium subsp.paratuberculosis). Conclusions Our findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA. PMID:24194951

Radomski, Nicolas; Kreitmann, Louis; McIntosh, Fiona; Behr, Marcel A.

2013-01-01

50

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

E-print Network

this compound as part of the DNA extraction process. We also describe a method for demonstrating the presence to inhibit PCR (for an excellent review see [4]), but those commonly co-extracted with aDNA from teeth, bones- dicated by a discolored DNA extract, usually tinted yellowish- to reddish-brown [13,28,29]. Inhibited DNA

Kemp, Brian M.

51

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

52

Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling  

PubMed Central

Background Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Results Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. Conclusions This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS. PMID:24980254

2014-01-01

53

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

2011-09-01

54

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

55

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

PubMed

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

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

2011-11-01

56

The effect of extraction temperature, time and number of steps on the antioxidant capacity of methanolic banana peel extracts  

Microsoft Academic Search

A solvent extraction method was developed to obtain methanolic extracts rich in antioxidants from banana peel. Central composite design 23+star and response surface methodology were used in order to optimise the number of extraction steps, extraction temperature and extraction time. The number of extractions was statistically the most significant factor in scavenging activity against both DPPH and ABTS+ radicals and

Rafaela Gonzlez-Montelongo; M. Gloria Lobo; Mnica Gonzlez

2010-01-01

57

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

58

Forensic animal DNA analysis using economical two-step direct PCR.  

PubMed

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 180.84 ng/?L. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire() Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community. PMID:24435950

Kitpipit, Thitika; Chotigeat, Wilaiwan; Linacre, Adrian; Thanakiatkrai, Phuvadol

2014-03-01

59

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. PMID:23461534

Li, Yan; Araki, Hiroyuki

2013-01-01

60

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

PubMed Central

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the vant Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

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

2014-01-01

61

DNA extraction from vegetative tissue for next-generation sequencing.  

PubMed

The quality of extracted DNA is crucial for several applications in molecular biology. If the DNA is to be used for next-generation sequencing (NGS), then microgram quantities of good-quality DNA is required. In addition, the DNA must substantially be of high molecular weight so that it can be used for library preparation and NGS sequencing. Contaminating phenol or starch in the isolated DNA can be easily removed by filtration through kit-based cartridges. In this chapter we describe a simple two-reagent DNA extraction protocol which yields a high quality and quantity of DNA which can be used for different applications including NGS. PMID:24243190

Furtado, Agnelo

2014-01-01

62

A fully automatable enzymatic method for DNA extraction from plant tissues  

PubMed Central

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

Manen, Jean-Francois; Sinitsyna, Olga; Aeschbach, Lorene; Markov, Alexander V; Sinitsyn, Arkady

2005-01-01

63

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

64

A rapid DNA extraction method suitable for human papillomavirus detection.  

PubMed

Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples. PMID:24443320

Brestovac, Brian; Wong, Michelle E; Costantino, Paul S; Groth, David

2014-04-01

65

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

2013-01-01

66

An optimized DNA extraction protocol for benthic Didymosphenia geminata.  

PubMed

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

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

2014-09-01

67

Extraction of DNA from the plant Kalancho daigremontiana.  

PubMed

This protocol describes how to isolate genomic DNA from leaves and stems of Kalancho daigremontiana. The procedure can be applied to adult leaves, but it is best to use younger leaves because they have fewer secondary metabolites and polysaccharides, which can interfere with the DNA extraction. The resulting DNA can be used for polymerase chain reactions (PCRs), Southern blots, or other applications. PMID:20147049

Garcs, Helena; Sinha, Neelima

2009-10-01

68

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. PMID:20011662

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

69

A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis.  

PubMed

As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA. PMID:12664992

Miller Coyle, Heather; Shutler, Gary; Abrams, Sharon; Hanniman, Janet; Neylon, Suzanne; Ladd, Carll; Palmbach, Timothy; Lee, Henry C

2003-03-01

70

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

PubMed

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

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

2013-09-01

71

A protocol for high-quality genomic DNA extraction from legumes.  

PubMed

Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/?L with an average of 1459 ng/?L was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US$2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species. PMID:23079974

Agbagwa, I O; Datta, S; Patil, P G; Singh, P; Nadarajan, N

2012-01-01

72

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

S, Olga; Pereira, Jos Alberto; Baptista, Paula

2011-01-01

73

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

PubMed Central

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 compoundsnamely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 78.0 ?g/?L and the purity, evaluated by the ratio A260/A280, was 1.80 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR. PMID:21747730

Sa, Olga; Pereira, Jose Alberto; Baptista, Paula

2011-01-01

74

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

PubMed Central

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

Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-01-01

75

The influence of substrate on DNA transfer and extraction efficiency.  

PubMed

The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15?L of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (p<0.05) differences also exist among the DNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (p<0.05) loss of DNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and concentration efficiencies among substrates is proposed. PMID:23040243

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

2013-01-01

76

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

E-print Network

Strawberry DNA Extraction 2009 1 Minority Science Programs ­ School regular household chemicals to extract DNA from strawberries and other fruits. Each chemical has strand. #12;Strawberry DNA Extraction 2009 2 Minority Science Programs ­ School

Rose, Michael R.

77

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

2012-09-01

78

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

79

Crowdsourcing step-by-step information extraction to enhance existing how-to videos  

E-print Network

Millions of learners today use how-to videos to master new skills in a variety of domains. But browsing such videos is often tedious and inefficient because video player interfaces are not optimized for the unique step-by-step ...

Nguyen, Phu Tran

80

A high-throughput DNA extraction method for barley seed  

Microsoft Academic Search

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

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

2003-01-01

81

Novel method of DNA extraction from bones assisted DNA identification of World Trade Center victims  

Microsoft Academic Search

DNA identification of many mass fatality victims of the 11 September 2001 attack on the World Trade Centers (WTC) in New York City required development of new analytical methods. Development of novel STR multiplex sets with improved performance using challenged DNA samples is described in an accompanying paper. Here we describe modifications and improvements to procedures used to extract DNA

T Bille; R Wingrove; M Holland; C Holland; C Cave; J Schumm

2004-01-01

82

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

2006-04-01

83

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, Sana; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-01-01

84

An inexpensive and rapid method for extracting papilionoid genomic DNA from herbarium specimens.  

PubMed

Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols. PMID:20645258

Riahi, M; Zarre, S; Maassoumi, A A; Attar, F; Kazempour Osaloo, S

2010-01-01

85

Extracting biological knowledge from DNA sequences  

SciTech Connect

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

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

1996-12-31

86

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

PubMed Central

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

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

2013-01-01

87

Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.  

PubMed

Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms. PMID:25053969

Healey, Adam; Furtado, Agnelo; Cooper, Tal; Henry, Robert J

2014-01-01

88

Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species  

PubMed Central

Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms. PMID:25053969

2014-01-01

89

Fern spore extracts can damage DNA  

Microsoft Academic Search

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.

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

2000-01-01

90

Application of chemometric tools for automatic classification and profile extraction of DNA samples in forensic tasks.  

PubMed

In this paper a method for the automatic DNA spots classification and extraction of profiles associated in DNA polyacrylamide gel electrophoresis is presented and it integrates the use of image processing techniques and chemometrics tools. A software which implements this method was developed; for feature extraction a combination of a PCA analysis and a C4.5 decision tree were used. To obtain good results in the profile extraction only DNA spots are useful; therefore, it was necessary to solve a two-class classification problem among DNA spots and no-DNA spots. In order to perform the classification process with high velocity, effectiveness and robustness, comparative classification studies among support vector machine (SVM), K-NN and PLS-DA classifiers were made. The best results obtained with the SVM classifier demonstrated the advantages attributed to it in the literature as a two-class classifier. A Sequential Cluster Leader Algorithm and another one developed for the restoration of pattern missing spots were needed to conclude the profiles extraction step. The experimental results show that this method has a very effective computational behavior and effectiveness, and provide a very useful tool to decrease the time and increase the quality of the specialist responses. PMID:17605982

Talavera Bustamante, Isneri; Silva Mata, Francisco; Hernndez Gonzlez, Noslen; Gonzlez Gazapo, Ricardo; Palau, Juan; Ferreira, Marcia M Castro

2007-07-01

91

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR  

USGS Publications Warehouse

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W., III; Lindquist, H.D. Alan

2010-01-01

92

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

93

Propeller-Twisting of Base-pairs and the Conformational Mobility of Dinucleotide Steps in DNA  

Microsoft Academic Search

When DNA is bent around a protein, it must distort. The distortion occurs by changes in the conformation of successive dinucleotide steps. Bending does not necessarily occur uniformly: some steps might remain particularly rigid, i.e. they might deform relatively little, while others might take more than their proportional share of deformation. We investigate here the deformational capacity of specific dinucleotide

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

1996-01-01

94

DNA extraction and fingerprinting of commercial rice cereal products  

Microsoft Academic Search

DNA was extracted from commercial rice cereal products using modified conventional methods (CTAB, SDS and a commercial kit) in large fragments (>3kb) and with relatively high yields (1.410.7?g DNA per g of sample) and was used as template for the amplification of a single copy rice gene (i.e. MIPS) fragment (ca. 850bp) and microsatellite DNAs (ca. 120400bp). The cereal products

Xueliang Ren; Xiaoyang Zhu; Maarten Warndorff; Peter Bucheli; Qingyao Shu

2006-01-01

95

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

E-print Network

DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps GUDRUN DITTRICH and Entomology, University of Pretoria, Pretoria 0002, South Africa, ARC-Plant Protection Research Institute from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline

96

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA.  

PubMed

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis. PMID:17673324

Streitner, Nadine; Voss, Carsten; Flaschel, Erwin

2007-08-31

97

A simple method for extracting DNA from old skeletal material.  

PubMed

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents. PMID:7557753

Cattaneo, C; Smillie, D M; Gelsthorpe, K; Piccinini, A; Gelsthorpe, A R; Sokol, R J

1995-07-28

98

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

Microsoft Academic Search

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

Kirsty Phillips; Nicola McCallum; Lindsey Welch

99

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; Martn-Platero, Antonio Manuel; Reboleiro-Rivas, Patricia; Gonzlez-Lpez, Jess; Pozo, Clementina

2011-09-15

100

A method suitable for DNA extraction from humus-rich soil.  

PubMed

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils. PMID:24980851

Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

2014-11-01

101

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

102

Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells  

PubMed Central

Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells. PMID:23773807

Badralmaa, Yunden; Natarajan, Ven

2013-01-01

103

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

Microsoft Academic Search

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

Chirayu Desai; Datta Madamwar

2007-01-01

104

CSP-DNA EXTRACTION & STAGING LABORATORY Prices 2013 & 2014  

Cancer.gov

09/18/14 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

105

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

106

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.

107

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

PubMed

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

Hawash, Yousry

2014-06-01

108

A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants.  

PubMed

Protocols for genomic DNA extraction from plants are generally lengthy, since they require that tissues be ground in liquid nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening projects. PMID:18781397

Kotchoni, Simeon O; Gachomo, Emma W

2009-07-01

109

Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.  

PubMed

A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood. PMID:21041388

Springer, Nathan M

2010-11-01

110

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae  

Microsoft Academic Search

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality.\\u000a The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260\\/280 absorbance ratio. The total\\u000a DNA yield was measured by a fluorometer.

Lenka Drbkov; Jan Kirschner; ?estmr Vl?ek

2002-01-01

111

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

E-print Network

DNA extraction protocol involves suspend- ing macerated tissue in a buffer containing a detergent fungal cultures and sporocarps; single-copy microsatellite amplification from cultures for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts

California at Berkeley, University of

112

Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm.  

PubMed

The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes. PMID:18228540

Belefant-Miller, Helen; Ledbetter, Cindy; Bennett, Selester

2008-03-01

113

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

E-print Network

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

114

Optimization of DNA Extractions from Iron-rich Microbial Mats  

NASA Astrophysics Data System (ADS)

Iron is the fourth most abundant element in the Earth's crust and is potentially one of the most abundant energy sources on the earth as an electron donor for chemolithoautotrophicgrowth coupled to Fe(II) oxidation. Many microbes have adapted to this energy source. One such bacterial class are the Zetaproteobacteria, which dominate Iron-rich microbial mats at Loihi seamount. Although cell counts are very high (up to 5.3x108 cells/ml), efficient DNA yields are low in comparison. In this study we compared extraction efficiency across different methods and with the addition of various buffers. Regardless of protocol (i.e., kit), the addition of sodium citrate drastically increased the DNA yield. The addition of sodium citrate did not alter community structure as determined by T-RFLP and qPCR. Citrate is a well-known ferric iron chelator and will bind ferrous as well. The chelated iron is then unable to participate in the Fenton reaction and this stops the generation of hydroxyl radicals which in turn can react and degrade the extracted DNA. We have utilized this relationship to allow us to obtain nearly an order of magnitude more microbial community DNA per sample, which should also have implications when processing low biomass samples, e.g., from the deep subsurface.

Fullerton, H.; Hilton, T. S.; Moyer, C. L.

2013-12-01

115

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

116

DNA extraction from dried Schistosoma haematobium eggs isolated on nylon filters.  

PubMed

Genetic studies on Schistosoma haematobium are often carried out on DNA extracted from miracidia, cercariae or adult worms. This paper presents a method for extracting DNA from S. haematobium eggs collected from urine samples and stored on nylon filters at room temperature. DNA was extracted from dried S. haematobium eggs using the DNeasy Blood & Tissue Kit (QIAGEN Sample & Assay Technologies, Copenhagen, Denmark). Selected genes were amplified using PCR to verify that DNA extraction had been successful. DNA was extracted from 45 samples and 31 had a positive PCR reaction for either or both of the two selected genes. PMID:22381628

Reinstrup, L; Jrgensen, A; Vennervald, B J; Kristensen, T K

2012-04-01

117

Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100  

Microsoft Academic Search

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps.\\u000a We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient\\u000a PCR-based detection of transgenes from a variety

Kwon HwangBo; Su Hyun Son; Jong Suk Lee; Sung Ran Min; Suk Min Ko; Jang R. Liu; Dongsu Choi; Won Joong Jeong

2010-01-01

118

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

PubMed Central

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

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

119

Preparation of DNA extracted from environmental water samples for PCR amplification 1 Contribution number 268 from the Center of Marine Biotechnology. 1  

Microsoft Academic Search

Sephadex G-200 spun columns have been used to purify DNA extracted from aquatic samples. Nucleic acid recovery using a previously-described protocol was only 10 to 15%. We optimized this method by employing a high salt (0.2 M NaCl) TE buffer (pH 8.0) and four slow-speed centrifugation steps (130g) in a swing-out centrifuge. DNA recovery improved to approximately 75%. Purified DNA

Victoria M Boccuzzi; William L. Straube; Jacques Ravel; Rita R Colwell; Russell T Hill

1998-01-01

120

One-step extraction\\/methylation method for determining the fatty acid composition of processed foods  

Microsoft Academic Search

In a one-step extraction\\/methylation (OSM) method for determining individual fatty acids (FA) in processed food products,\\u000a freeze-dried samples, containing 1050 mg fat, were transmethylated without prior fat extraction with a mixture of methanol-HCl\\/toluene.\\u000a After washing the organic phase the formed FA methyl esters were ready for separation by gas-liquid chromatography (GLC).\\u000a The relative standard deviation for total FA content was

Franz Ulberth; Monika Henninger

1992-01-01

121

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

2013-01-01

122

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

PubMed

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories. PMID:20171997

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

2010-05-01

123

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

PubMed

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

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

2014-11-01

124

Deparaffinization With Mineral Oil: A Simple Procedure for Extraction of High-quality DNA from Archival Formalin-fixed Paraffin-embedded Samples.  

PubMed

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

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

2014-09-01

125

[Study of DNA extraction methods for testing for genetically modified organisms in soyproducts].  

PubMed

In order to evaluate three different methods for DNA extraction (CTAB, DNeasy Plant Mini Kit and Wizard DNA Clean-up Resin system), the yields of DNA extracted from soyproducts and the copy numbers of lectin genes amplified by quantitative PCR were compared. Fermented foods, such as miso and nattou, gave poor yields of DNA and low copy numbers with any method. However atsu-age and kinugoshi-tofu gave high-quality results with all methods. Kinako gave a high yield of DNA, but poor amplification. Boiled soybeans and soymilk showed in poor amplification. It is important to choose the appropriate DNA extraction method for each product. PMID:19029786

Moriuchi, Rie; Monma, Kimio; Kamata, Kunihiro; Ibe, Akihiro

2008-01-01

126

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

Microsoft Academic Search

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

Jason Baumsteiger; Jacob L. Kerby

2009-01-01

127

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

128

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

E-print Network

DNA BARCODING Tissue-direct PCR, a rapid and extraction-free method for barcoding of ferns F-W. LI gametophytes and young sporophytes often provide too little material for DNA extraction and are particularly distribution and population structure. Keywords: DNA barcoding, ferns, gametophytes, Tissue-direct PCR, young

129

Nuclear Assembly with k DNA in Fractionated Xenopus Egg Extracts: An Unexpected Role for Glycogen in  

E-print Network

Nuclear Assembly with k DNA in Fractionated Xenopus Egg Extracts: An Unexpected Role for Glycogen. Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template

Forbes, Douglass

130

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

E-print Network

A more consistent method for extracting and amplifying DNA from bee wings Elaine M. GOULD, Michelle reaction (PCR) amplification. Here, we describe an improved method for extracting DNA from bee wings using% amplification in subsequent PCR. DNA / bee wing / PCR / bashing beads / Chelex® 100 1. INTRODUCTION In recent

131

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

Microsoft Academic Search

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

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

1990-01-01

132

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

133

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics  

PubMed Central

Background DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequencesmitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. Methodology We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processoran automated high throughput DNA extraction systemand tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. Results The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183. Conclusions The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding. PMID:25415202

Vidergar, Nina; Toplak, Nataa; Kuntner, Matja

2014-01-01

134

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

Microsoft Academic Search

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

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

1994-01-01

135

Genomic DNA extraction protocols for bone samples: The comparison of Qiagen and Zymo Research spin columns  

Microsoft Academic Search

The aim of this study was to develop an extraction protocol for bone samples based on ZR Genomic DNA Tissue MicroPrep kit and perform a quantitative comparison with the existing silica extraction protocol based on Qiagen columns and evaluate the effect of demineralization on the quantity of extracted DNA.

Daniel Vanek; Marcela Silerova; Vladislava Urbanova; Lenka Saskova; Jitka Dubska; Michal Beran

136

Comparative study of DNA extraction methods for soybean derived food products  

Microsoft Academic Search

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

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

2008-01-01

137

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/l, 9.5pg/l and 0.4pg/l 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/l (115.5) and 690.1pg/l (207.1), while concentrations with addition of carrier RNA were 49414.5pg/l (9370.6) and 20982.7pg/l (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. PMID:23071624

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

2012-01-01

138

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

PubMed Central

Background Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Methods Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample students t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment. PMID:25067909

2014-01-01

139

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues.  

PubMed

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR. PMID:16723190

Rivero, Elena R C; Neves, Adriana C; Silva-Valenzuela, Maria G; Sousa, Suzana O M; Nunes, Fabio D

2006-01-01

140

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 A???/A??? 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-Lpez, C Y; Gonzlez-Mendoza, D; Grimaldo-Juarez, O

2013-01-01

141

Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy.  

PubMed

The authors report a case of Duchenne muscular dystrophy in which a deletion was determined by multiplex polymerase chain reaction using postmortem tissue from a proband who died in 1985, 2 years before the cloning of the dystrophin gene. Several extraction methods were analyzed to determine optimal conditions for recovery of DNA from fixed tissue. Variables examined were tissue lysis times and temperatures, a simple salting-out procedure for purification of DNA, polymerase chain reaction amplification of crude lysate versus purified DNA, and lysis of different tissues and tissue quantities. Extracted DNA was analyzed spectrophotometrically, electrophoretically, and by its suitability for polymerase chain reaction amplification using exon flanking primers of the dystrophin gene. Lysis at 37 degrees C for less than 24 hours led to the recovery of predominantly low-molecular-weight DNA, which was unsuitable for polymerase chain reaction in this experience. Lysis at 54 degrees C consistently yielded visible, spoolable quantities of high-molecular-weight DNA in as little as 24 hours. Direct amplification of crude, unpurified lysates was unsuccessful. However, purified samples yielded consistent amplification products. The purification step was simplified by substituting a rapid salting-out procedure for organic extractions. PMID:1615933

Forsthoefel, K F; Papp, A C; Snyder, P J; Prior, T W

1992-07-01

142

DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.  

PubMed Central

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo. Images PMID:1579437

Mahbubani, H M; Paull, T; Elder, J K; Blow, J J

1992-01-01

143

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

144

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

E-print Network

gently by inversion until the DNA has precipitated out of solution. Incubate at room temperature for 5Extraction of Genomic DNA ­ CTAB-Lysozyme Method Isolation of genomic DNA is necessary for PCR and Southern analysis. The CTAB- Lysozyme method of genomic DNA extraction yields good quality DNA. Typically

145

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

146

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues  

Microsoft Academic Search

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenolchloroform extraction method and with

Elena R. C. Rivero; Adriana C. Neves; Maria G. Silva-Valenzuela; Suzana O. M. Sousa; Fabio D. Nunes

2006-01-01

147

Optimisation of DNA extraction for AFLP analysis of mycorrhizal fungi of terrestrial orchids caladeniinae and drakaeinae  

Microsoft Academic Search

Published DNA extraction methods present a number of problems when applied to mycorrhizal fungi of native Australian terrestrial\\u000a orchids. Grinding with liquid nitrogen shears the DNA, and other pulverisation methods yield too little DNA. We found that\\u000a freezing the fungal sample with liquid nitrogen, with no grinding, followed by the Qiagen DNeasy extraction procedure produced\\u000a good yields of high-molecular-weight DNA.

Penelope S. Hollick; Robyn J. Taylor; Jen A. Mccomb; Kingsley W. Dixon; Siegfried L. Krauss

2004-01-01

148

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

2012-01-01

149

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar  

PubMed Central

Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA SPIN Kit for Soil (FD kit), the PowerSoil DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.

2014-01-01

150

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT  

PubMed Central

O6-alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in clinical trials as anticancer therapeutics. Computational approaches introduced recently for harvesting and statistically characterizing trajectories of molecularly rare events now enable us to elucidate a pathway for nucleotide flipping by AGT and the forces that promote it. In contrast to previously proposed flipping mechanisms, we observe a two-step process that promotes a kinetic, rather than a thermodynamic, gate-keeping strategy for lesion discrimination. Connection is made to recent single-molecule studies of DNA-repair proteins sliding on DNA to understand how they sense subtle chemical differences between bases efficiently. PMID:18353991

Hu, Jie; Ma, Ao; Dinner, Aaron R.

2008-01-01

151

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

Microsoft Academic Search

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube,\\u000a one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions.\\u000a The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200300 individuals

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

2001-01-01

152

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

NASA Astrophysics Data System (ADS)

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

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

2010-06-01

153

A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis.  

PubMed

The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir. PMID:8621577

Wildung, M R; Croteau, R

1996-04-19

154

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.  

PubMed Central

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs. PMID:7760792

Gilbert, D M; Miyazawa, H; DePamphilis, M L

1995-01-01

155

Telomere length varies by DNA extraction method: Implications for epidemiologic research  

PubMed Central

Background Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from colorectal cancer patients. Methods We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 colorectal cancer patients and 2,952 healthy controls. DNA was extracted with Phenol/Chloroform, PureGene or QIAamp. Results We observed differences in RTL depending on DNA extraction method (p<0.001). Phenol/Chloroform extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01-6.54) ) compared to PureGene extracted DNA (mean RTL of 0.75; range 0.00-12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02-3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17-0.58-) was smaller than from either PureGene or Phenol/Chloroform (ranges:0.04-2.67 and 0.32-2.81, respectively). Conclusions RTL measured by qPCR from QIAamp-extracted DNA was smaller than from either PureGene or Phenol/Chloroform (p<0.001). Impact Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. PMID:24019396

Cunningham, Julie M.; Johnson, Ruth A.; Litzelman, Kristin; Skinner, Halcyon G.; Seo, Songwon; Engelman, Corinne D.; Vanderboom, Russell J.; Kimmel, Grace W.; Gangnon, Ronald E.; Riegert-Johnson, Douglas L.; Baron, John A.; Potter, John D.; Haile, Robert; Buchanan, Daniel D.; Jenkins, Mark A.; Rider, David N.; Thibodeau, Stephen N.; Petersen, Gloria M.; Boardman, Lisa A.

2013-01-01

156

Limitations and recommendations for successful DNA extraction from forensic soil samples: a review.  

PubMed

Soil is commonly used in forensic casework to provide discriminatory power to link a suspect to a crime scene. Standard analyses examine the intrinsic properties of soils, including mineralogy, geophysics, texture and colour; however, soils can also support a vast amount of organisms, which can be examined using DNA fingerprinting techniques. Many previous genetic analyses have relied on patterns of fragment length variation produced by amplification of unidentified taxa in the soil extract. In contrast, the development of advanced DNA sequencing technologies now provides the ability to generate a detailed picture of soil microbial communities and the taxa present, allowing for improved discrimination between samples. However, DNA must be efficiently extracted from the complex soil matrix to achieve accurate and reproducible DNA sequencing results, and extraction efficacy is highly dependent on the soil type and method used. As a result, a consideration of soil properties is important when estimating the likelihood of successful DNA extraction. This would include a basic understanding of soil components, their interactions with DNA molecules and the factors that affect such interactions. This review highlights some important considerations required prior to DNA extraction and discusses the use of common chemical reagents in soil DNA extraction protocols to achieve maximum efficacy. Together, the information presented here is designed to facilitate informed decisions about the most appropriate sampling and extraction methodology, relevant both to the soil type and the details of a specific forensic case, to ensure sufficient DNA yield and enable successful analysis. PMID:24796953

Young, Jennifer M; Rawlence, Nicolas J; Weyrich, Laura S; Cooper, Alan

2014-05-01

157

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

PubMed

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

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

2014-01-01

158

Comparison of DNA extraction methods for sweet corn and processed sweet corns.  

PubMed

DNA was extracted from sweet corn and its processed products using four DNA extraction methods: the CTAB method, the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G. DNA was successfully extracted from raw sweet corn and baby corn samples using all four methods. Meanwhile, from frozen, canned, and dry pack products, DNA was well extracted using the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G, but not enough with the CTAB method. The highest yield of DNA was obtained with Genomic-tip 20/G. The degree of degradation of extracted DNA was observed to increase in the order of raw, frozen, canned, dry pack, and baby corn samples. To evaluate the quality of extracted DNA, real-time PCR analyses were conducted using three maize endogenous genes. The DNAs extracted using GM Quicker 3 had high purity, suggesting that GM Quicker 3 would be the most suitable method for DNA extraction from processed sweet corn products. PMID:24025210

Takabatake, Reona; Noritake, Hiromichi; Noguchi, Akio; Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Teshima, Reiko; Mano, Junichi; Kitta, Kazumi

2013-01-01

159

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...

160

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

161

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...

162

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

EPA Science Inventory

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

163

Extracting Strawberry DNA Adapted from http://www.genome.gov/Pages/Education/Modules/StrawberryExtractionInstructions.pdf  

E-print Network

3 (not allergic to strawberries): Put coffee filter over cup. Pour crushed strawberries into filterExtracting Strawberry DNA Adapted from http://www.genome.gov/Pages/Education/Modules/StrawberryExtractionInstructions.pdf for a group of 5 students with an adult moderator Strawberries have enormous genomes. Humans have two copies

Gruber, Jonathan

164

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

E-print Network

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

Michel Jr., Frederick C.

165

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

Microsoft Academic Search

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

Tae-Jin Kang; Moon-Sik Yang

2004-01-01

166

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

E-print Network

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

167

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

168

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

PubMed

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

Gong, Rui; Li, Shengying

2014-01-01

169

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

PubMed

According to the French legislation on medical biology (January 16th, 2010), all biological laboratories must be accredited according to ISO15189 for at least 50% of their activities before the end of 2016. The extraction of DNA from a sample of interest, whether solid or liquid is one of the critical steps in molecular biology and specifically in somatic or constitutional genetic. The extracted DNA must meet a number of criteria such quality and also be in sufficient concentration to allow molecular biology assays such as the detection of somatic mutations. This paper describes the validation of the extraction and purification of DNA using chromatographic column extraction and quantitative determination by spectrophotometric assay, according to ISO15189 and the accreditation technical guide in Human Health SH-GTA-04. PMID:24113450

Harl, Alexandre; Lion, Mava; Husson, Marie; Dubois, Cindy; Merlin, Jean-Louis

2013-01-01

170

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

2007-03-01

171

DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods.  

PubMed

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa-stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze-thaw method, boiling in 10% Chelex-100 resin solution, proteinase K/Tween 20/NP-40 method coupled with simplified phenol/ chloroform/isoamyl alcohol protocol or salting-out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen. Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa-stained bone marrow slides. PMID:9609534

Vince, A; Poljak, M; Seme, K

1998-05-01

172

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

173

Tailing DNA aptamers with a functional protein by two-step enzymatic reaction.  

PubMed

An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 3'-OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins. PMID:23806788

Takahara, Mari; Hayashi, Kounosuke; Goto, Masahiro; Kamiya, Noriho

2013-12-01

174

Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device.  

PubMed

A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification. PMID:19904411

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

2009-12-01

175

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. PMID:10621838

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

1999-01-01

176

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).  

PubMed

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ? 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. PMID:22414329

Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

2012-05-01

177

Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction.  

PubMed

Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 ?g/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction. PMID:24280193

Sagar, Kalpana; Singh, Salam Pradeep; Goutam, Kapil Kumar; Konwar, Bolin Kumar

2014-02-01

178

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

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

179

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

PubMed

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

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

2014-01-01

180

Elimination of bioweapons agents from forensic samples during extraction of human DNA.  

PubMed

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1h compromised the ability to complete genetic profiling. PMID:25069670

Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

2014-11-01

181

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.  

PubMed

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates. PMID:24916950

Vorsters, A; Van den Bergh, J; Micalessi, I; Biesmans, S; Bogers, J; Hens, A; De Coster, I; Ieven, M; Van Damme, P

2014-11-01

182

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

PubMed Central

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

Avanesyan, Alina

2014-01-01

183

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens  

PubMed Central

Background The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean Fecal DNA Isolation Kit, M; QIAamp DNA Stool Mini Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Method Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Results Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. Conclusion We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities. PMID:20492702

2010-01-01

184

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

PubMed

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

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

2014-06-01

185

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

PubMed

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 (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. PMID:25213972

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

2011-06-15

186

Comparative Analysis of Different DNA Extraction Protocols: A Fast, Universal Maxi-Preparation of High Quality Plant DNA for Genetic Evaluation and Phylogenetic Studies  

Microsoft Academic Search

Four DNA extraction protocols were compared for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and

U. M. Csaikl; H. Bastian; R. Brettschneider; S. Gauch; A. Meir; M. Schauerte; F. Scholz; C. Sperisen; B. Vornam; B. Ziegenhagen

1998-01-01

187

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. PMID:9023118

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

1997-01-01

188

Direct DNA extraction for PCR-mediated assays of soil organisms.  

PubMed

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 losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis. PMID:8526511

Volossiouk, T; Robb, E J; Nazar, R N

1995-11-01

189

A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS.  

PubMed

Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method. PMID:21924387

Sekikawa, Takahiro; Kawasaki, Yu; Katayama, Yasuto; Iwahori, Keisuke

2011-12-15

190

Echinococcus granulosus: DNA Extraction from Germinal Layers Allows Strain Determination in Fertile and Nonfertile Hydatid Cysts  

Microsoft Academic Search

Kamenetzky, L., Canova, S. G., Guarnera, E. A., and Rosenzvit, M. C. 2000. Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts. Experimental Parasitology95, 122127. A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide\\/chloroform extraction and an adsorption to

Laura Kamenetzky; Sergio G. Canova; Eduardo A. Guarnera; Mara C. Rosenzvit

2000-01-01

191

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

Microsoft Academic Search

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

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

1994-01-01

192

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 (46C) 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. PMID:24294115

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

2013-01-01

193

[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

194

Comparison of six extraction techniques for isolation of DNA from filamentous fungi.  

PubMed

Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures. PMID:10075499

van Burik, J A; Schreckhise, R W; White, T C; Bowden, R A; Myerson, D

1998-10-01

195

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

196

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

PubMed Central

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

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

2014-01-01

197

Extraction of high-quality genomic DNA from latex-containing plants  

Microsoft Academic Search

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA.

An Michiels; Wim Van den Ende; Mark Tucker; Liesbet Van Riet; Andr Van Laere

2003-01-01

198

Methodological comparison of DNA extraction from Holcocerrus hippophaecolus (Lepidoptera: Cossidae) for AFLP analysis  

Microsoft Academic Search

Amplified fragment length polymorphism (AFLP) is a powerful DNA fingerprinting technique for studying genetic relationships\\u000a and genetic diversity in insects. However, the crucial prerequisite for AFLP analysis is to extract DNA of high quality. In\\u000a this study, we evaluate four different protocols (SDS method, improved SDS method, CTAB method and a complex method with SDS\\u000a and CTAB) for isolating DNA

Min Chen; Yang-yu Zhu; Jing Tao; You-qing Luo

2008-01-01

199

DOI: 10.1002/cbic.201200167 Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage  

E-print Network

DOI: 10.1002/cbic.201200167 Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage of a polymer linkage that polymerase enzymes cannot read, thereby limiting the accessibility and utility of ex mode at higher substrate concentrations; and 5) produces a linkage that can be tolerated in a template

Williams, Loren

200

Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing.  

PubMed

Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected. PMID:22230933

Rittenour, William R; Park, Ju-Hyeong; Cox-Ganser, Jean M; Beezhold, Donald H; Green, Brett J

2012-03-01

201

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 g 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

202

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.  

PubMed

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods. PMID:19714982

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

203

Replication of colicin E1 plasmid DNA added to cell extracts.  

PubMed Central

Closed-circular DNA of colicin E1 plasmid can undergo a round of semiconservative replication when added to an extract of Escherichia coli. Extracts of cells that do not carry the plasmid are able to perform complete replication of the plasmid. Replication requires de novo RNA synthesis but not protein synthesis. PMID:1093162

Tomizawa, J I; Sakakibara, Y; Kakefuda, T

1975-01-01

204

Direct Extraction of DNA from Soils for Studies in Microbial Ecology  

Microsoft Academic Search

Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be

Mark A. Schneegurt; Sophia Y. Dore; Charles F. Kulpa

205

Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water.  

PubMed

Giardia lamblia is one of the most recognized waterborne protozoan parasites causing gastrointestinal disease. A simple but effective DNA extraction protocol for real-time PCR detection from surface water samples was developed in this study. Eleven protocols were compared, which consisted of freeze-thaw treatments (liquid N(2) and boiling water) and purification using the Qiagen DNeasy kit, together with different combinations of proteinase K, PVP360, GITC and Chelex 100 incubation. Using concentrated surface water samples spiked with G. lamblia cysts, the necessary steps for high DNA recovery were shown to be freeze-thaw, DNeasy purification and Chelex 100 incubation. Multiple rounds of freeze-thaw treatment (five cycles per round) were reported for the first time in this study to significantly increase the DNA yield from G. lamblia cysts, from ~20% after one round of freeze-thaw to 40 and 70% after two and three-rounds of freeze-thaw, respectively. More than three rounds of freeze-thaw treatment did not promote additional DNA recovery. The final protocol included three-three-rounds of freeze-thaw treatment, DNeasy purification and Chelex 100 incubation. This method was simpler, more cost-effective, and had a comparable DNA recovery to methods involving immunomagnetic separation. PMID:19499328

Yu, Xin; Van Dyke, Michele I; Portt, Andrea; Huck, Peter M

2009-08-01

206

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

PubMed Central

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

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

1985-01-01

207

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

USGS Publications Warehouse

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

Baker, Erin J.; Kellogg, Christina A.

2014-01-01

208

An efficient DNA extraction method for desert Calligonum species.  

PubMed

Genetic conservation programs in arid environments rely on molecular methods for diversity assessments. DNA-based molecular profiling will aid in conservation and protection of species from genetic erosion. Obtaining intact genomic DNA from Calligonum species, of sufficiently high-quality that is readily amplifiable using PCR, is challenging because of the presence of the exceptionally large amount of oxidized polyphenolic compounds, polysaccharides, and other secondary metabolites. The present method involves a modification of the available CTAB method employing higher concentrations of NaCl and CTAB, and incorporating PEG 6000 (1%) and glucose. The yield of DNA was 60-670 ?g g(-1) of fresh tissue. The protocol has been tested with two species from the arid region. The DNA isolated was successfully amplified by two ITS primer pairs. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region among and within Calligonum species followed by sequencing is under way. PMID:21681578

Abdellaoui, Raoudha; Gouja, Hassen; Sayah, Amel; Neffati, Mohamed

2011-12-01

209

BRCA2: One Small Step for DNA Repair, One Giant Protein Purified  

PubMed Central

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer. PMID:24348212

Jensen, Ryan B.

2013-01-01

210

Evaluation of methods for extracting Xylella fastidiosa DNA from the glassy-winged sharpshooter.  

PubMed

The recent spread of the plant pathogenic bacterium Xylclla fastidiosa Wells et al. by an invasive vector species, Homalodisca coagulata Say, in southern California has resulted in new epidemics of Pierce's disease of grapevine. Our goal is to develop an efficient method to detect low titers of X. fastidiosa in H. coagulata that is amenable to large sample sizes for epidemiological studies. Detection of the plant pathogenic bacterium X. fastidiosa in its insect vector is complicated by low titers of bacteria, difficulty in releasing it from the insect mouthparts and foregut, and the presence of substances in the insect that inhibit polymerase chain reaction (PCr). To select the optimal protocol for DNA extraction to be used with PCR, we compared three standard methods and 11 commercially available kits for relative efficiency of X. fastidiosa DNA extraction in the presence of insect tissue. All of the protocols tested were proficient at extracting DNA from pure bacterial culture (1 x 10(5) cells), and all but one protocol successfully extracted sufficient bacterial DNA in the presence of insect tissue. Three DNA extraction techniques, immunomagnetic separation, the DNeasy Tissue kit (Qiagen, Hercules, CA), and Genomic DNA Purification kit (Fermentus, Hanover, MD), were compared more closely using a dilution series of X. fastidiosa (5000-0 cells) with and without insect tissue present. The DNeasy Tissue kit was the best kit tested, allowing detection of 5 x 10(3) X. fastidiosa cells with an insect head background. PMID:15279249

Bextine, Blake; Tuan, Shu-Jen; Shaikh, Harris; Blua, Matthew; Miller, T A

2004-06-01

211

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

PubMed Central

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 specimen 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; O.D 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 MSP 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.

2012-01-01

212

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

PubMed

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

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

2010-01-01

213

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

PubMed Central

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

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

2014-01-01

214

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

Microsoft Academic Search

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

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

2003-01-01

215

Evaluating the efficacy of various thermo-stable polymerases against co-extracted PCR inhibitors in ancient DNA samples  

E-print Network

Evaluating the efficacy of various thermo-stable polymerases against co-extracted PCR inhibitors were evaluated for their efficacy against PCR inhibitors co-extracted with DNA from 63 ancient salmon potentially inhibit PCR and are routinely encountered in both the study of ancient DNA (aDNA) (see review

Kemp, Brian M.

216

Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification.  

PubMed

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products. PMID:21979091

Yang, Jing-Iong; Huang, Hsiao-Yun; Chou, Yii-Cheng; Chen, Chien-Cheng; Lee, Guo-Chi; Chang, Hsueh-Wei

2011-01-01

217

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

2006-09-18

218

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

PubMed Central

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

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

2014-01-01

219

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

Microsoft Academic Search

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

T. Sengl; S. Yurtseven; M. Cetin; A. Kocyigit; B. Sgt

220

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

2013-01-01

221

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

EPA Science Inventory

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

222

Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps  

PubMed Central

SUMMARY By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading strand synthesis taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound-copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without GC-dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate while the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a new model where the helicase and polymerase are moving in one-nucleotide steps and DNA synthesis drives fork unwinding and a role of the helicase is to trap the unwound bases and prevent DNA reannealing. PMID:24630996

Pandey, Manjula; Patel, Smita S.

2014-01-01

223

DNA extraction from paraffin embedded material for genetic and epigenetic analyses.  

PubMed

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures (1). To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion (2). Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA (3). DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization (4) (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples. PMID:21490570

Pikor, Larissa A; Enfield, Katey S S; Cameron, Heryet; Lam, Wan L

2011-01-01

224

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

PubMed

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

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

2014-01-01

225

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

PubMed Central

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

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

2014-01-01

226

DNA extraction from hair shafts of wild Brazilian felids and canids.  

PubMed

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the So Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme. PMID:21174262

Alberts, C C; Ribeiro-Paes, J T; Aranda-Selverio, G; Cursino-Santos, J R; Moreno-Cotulio, V R; Oliveira, A L D; Porchia, B F M M; Santos, W F; Souza, E B

2010-01-01

227

DNA Extraction from Nocardia Species for Special Genes Analysis Using PCR  

PubMed Central

Background: Nocardia species have a complex cell wall structure similar to that of mycobacteria, and the extraction of DNA from this bacterium is extremely difficult. Currently, to identify Nocardia species particularly, it is essential to utilize molecular techniques. Aims: In the present study, we investigated STET (sodium chloride-TRIS-EDTA-triton) buffer for the extraction of high-quality genomic DNA from 20 clinical and environmental isolates. Materials and Methods: The extracted DNA was evaluated for portion of the 16S rRNA, 65-kDa heat-shock protein and 16S rRNA genes via polymerase chain reaction. Results: The extracted DNA had high molecular mass, and its concentration and purity was suitable when tested in 1% agarose gel, and using UV spectrophotometry. Amplification of three different genes was successfully performed. Conclusion: This paper reveals an inexpensive, reproducible and efficient method of DNA extraction from Nocardia species, which is appropriate for accurate identification of this bacterium via polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism. PMID:24926450

Bafghi, Mehdi Fatahi; Eshraghi, Seyyed Saeed; Heidarieh, Parvin; Habibnia, Shadi; Nasab, Masoumeh Rasouli

2014-01-01

228

Molecular techniques: Extracting DNA from dried dots, PCR and sequencing  

E-print Network

blood dot is cut from a disk with a razor blade (single sharp edge). Each blade is used twice, once of the toxicity of the chemicals used. The kit protocol is followed except 50 µL of elution buffer or water is used at the end. If water is used to elute the DNA from the membrane, care is taken that the water

Schall, Joseph J.

229

Mechanistic pathway for controlled extraction of guest molecule bound to herring sperm DNA using ?-cyclodextrin  

NASA Astrophysics Data System (ADS)

trans-2-[4-(Dimethylamino)styryl]benzothiazole (DMASBT) is known to have dual emitting states where the locally excited (LE) state is responsible for fluorescence in less polar environment and in polar milieu fluorescence is from the twisted intramolecular charge transfer (TICT) state. This compound also undergoes minor groove binding to herring sperm DNA (hsDNA) evidenced by the absorption spectra before and after the binding process and an effect on DMASBT fluorescence by an anionic quencher. The binding occurs efficiently in a 1:1 manner, i.e. one guest molecule binds to one site on the hsDNA. Instead of following the DNA twist, the aromatic part seems to project outward. Thus, the bound molecule can be successfully extracted out from the DNA in a controlled way by the hydrophobic cavity of ?-cyclodextrin (?-CD). The extraction starts even with a low concentration of ?-CD and increases as the concentration is increased. Absorption, steady-state and time resolved fluorescence spectroscopic methods have been employed to explore the mechanistic pathway of binding of DMASBT to hsDNA. The mechanistic approach toward controlled extraction of the guest molecules from hsDNA by ?-CD is reported and is expected to serve a significant purpose in treatment of drug overdose.

Jaffer, S. Syed; Ghosh, Prasun; Purkayastha, Pradipta

2011-05-01

230

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. PMID:22521908

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

2012-01-01

231

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification.  

PubMed

The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species. PMID:24617319

Beroiz, B; Couso-Ferrer, F; Ortego, F; Chamorro, M J; Arteaga, C; Lombardero, M; Castaera, P; Hernndez-Crespo, P

2014-09-01

232

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

233

Validation of a DNA IQ-based extraction method for TECAN robotic liquid handling workstations for processing casework  

Microsoft Academic Search

A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ system was optimized and validated on TECAN Genesis 150\\/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead\\/DNA complex washes

Chantal J. Frgeau; C. Marc Lett; Ron M. Fourney

2010-01-01

234

The effects from DNA extraction methods on the evaluation of microbial diversity associated with human colonic tissue.  

PubMed

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host-microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account. PMID:21153634

Cuv, Praic; Aguirre de Crcer, Daniel; Jones, Michelle; Klaassens, Eline S; Worthley, Daniel L; Whitehall, Vicki L J; Kang, Seungha; McSweeney, Christopher S; Leggett, Barbara A; Morrison, Mark

2011-02-01

235

Experiments in DNA Extraction and PCR Amplification from Bighorn Sheep  

E-print Network

materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet extraction procedures relative to reliability of genotypes obtained. Our example involves bighorn sheep (Ovis canadensis), an herbivore for which blood and tissue samples are mostly difficult and costly to obtain

Epps, Clinton Wakefield

236

Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae).  

PubMed

Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/?L). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/?L) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/?L DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies. PMID:22653632

Borges, D B; Amorim, M B; Waldschmidt, A M; Mariano-Neto, E; Vivas, C V; Pereira, D G

2012-01-01

237

Photoreactive DNA probes as a tool for studying the translesion synthesis system in Mammalian cell extracts.  

PubMed

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies that has evolved to enable orga-nisms to replicate their genome despite the presence of unrepaired damage. TLS complexes are dynamic systems composed of DNA polymerases and associated protein factors. Therefore, it is hard to study these assembles by X-ray analysis or other instrumental methods. Here, we have suggested applying the photoaffinity labeling technique for studying the TLS system in nuclear/cellular extracts. As a tool we proposed to use partial DNA duplexes containing base-substituted photoreactive deoxynucleotides at the 3' end of primer opposite to DNA damage at the template strand. We demonstrated that photoreactive dNTPs can be potentially used to synthesize photoreactive DNA probes mimicking the DNA intermediates of the first stage of translesion synthesis by specialized DNA polymerases. We used synthetic apurinic/apyrimidinic site (AP-site) - tetrahydrofuran (THF) and 8 oxoguanine as damages in +1 position of the template strand with respect to 3' end of primer. Activity of human DNA polymerases beta and lambda was exploited for construction of photoreactive DNAs using photo derivatives of dNTPs. The kinetic parameters of the elongation reaction in model systems were estimated. Using photoaffinity crosslinking we found that only a few proteins in the bovine testis nuclear extract were strongly labeled by TLS probes. PMID:18336335

Belousova, Ekaterina A; Crespan, Emmanuele; Lebedeva, Natalia A; Rechkunova, Nadejda I; Hbscher, Ulrich; Magaand, Giovanni; Lavrik, Olga I

2008-03-01

238

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

239

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

PubMed

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types. PMID:17960375

Carrigg, Cora; Rice, Olivia; Kavanagh, Siobhn; Collins, Gavin; O'Flaherty, Vincent

2007-12-01

240

Inhibition of Taq DNA polymerase by seaweed extracts from British Columbia, Canada and Korea  

Microsoft Academic Search

Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the\\u000a presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity.\\u000a At the concentration of 5 g of methanol extract in 25L reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase,\\u000a one

Hyung-Joo Jin; Chul Hyun Sohn; R. E. DeWreede; G. H. N. Towers; J. B. Hudson

1997-01-01

241

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 0.3200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22118 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

242

DNA End-Joining Catalyzed by Human Cell-Free Extracts  

Microsoft Academic Search

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV\\/Xrcc4 and requires Ku70, Ku86,

Peter Baumann; Stephen C. West

1998-01-01

243

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. PMID:23868090

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

2013-01-01

244

Simple method of genomic DNA extraction from Rhizobia in dried nodules of Phaseolus vulgaris for strain differentiation by PCR-based DNA fingerprinting  

Microsoft Academic Search

Repetitive DNA peR fingerprinting of bacterial genomic DNA is a useful tool for typing and differentiation of rhizobial strains. The method was reported to be suitable for strain differentiation of Rhizobia present in individual root nodules of some leguminous plants without the need for isolation and cultivation of the strains, in which rhizobial genomic DNA was extracted directly from each

Choochad Santasup; Keishi Senoo; Ampan Bhromsiri; Arawan Shutsrirung; Akiyoshi Tanaka; Hitoshi Obata

2000-01-01

245

Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid.  

PubMed

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

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

2009-12-01

246

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

Microsoft Academic Search

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

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

2009-01-01

247

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

PubMed

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

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

2015-02-01

248

Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.  

PubMed

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

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

2011-05-01

249

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

PubMed

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

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

2014-01-01

250

Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.  

PubMed

Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al?(SO?)? were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al?(SO?)? for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

2014-02-01

251

Evaluation of MolYsis Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.  

PubMed

Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P<0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood. PMID:24503182

McCann, Chase D; Jordan, Jeanne A

2014-04-01

252

Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.  

PubMed

DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-?m sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56C. A second section was directly incubated at 120C and subsequently lysed at 65C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing. PMID:21704270

Steinau, Martin; Patel, Sonya S; Unger, Elizabeth R

2011-07-01

253

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

PubMed Central

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL(UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies. PMID:22952770

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

2012-01-01

254

Standardization of DNA extraction from methanol acetic acid fixed cytogenetic cells of cattle and buffalo.  

PubMed

The aim of the study is to standardize the simple method for extracting DNA from cells fixed in fixative (3:1 ratio of methanol and acetic acid glacial) mostly used for chromosomal studies in cattle and buffaloes. These fixed cells were stored for more than 6 months at refrigerated temperature. The fixed cells were washed 2-3 times by the ice cold 1x Phosphate Buffer Saline (PBS) with pH 7.4, so that effect of fixative may be eliminated. The genomic DNA was extracted by adding cell lysis and nucleus lysis buffers. The quality and quantity of DNA were estimated. The readings of nano drop and agarose gel electrophoresis indicate good quality DNA isolated with a rapid and simple protocol routinely using in our laboratory. The method enables us to study the DNA of a cattle and buffaloes after completing cytogenetic investigation or in cases where DNA samples are otherwise not available. This protocol may be useful for molecular analysis of DNA from fixed cells palettes. PMID:24506057

Kotikalapudi, Rosaiah; Patel, Rajesh K; Katragadda, Sanghamitra

2013-12-01

255

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

2011-10-13

256

Reactive Molecular Dynamics study on the first steps of DNA-damage by free hydroxyl radicals  

E-print Network

We employ a large scale molecular simulation based on bond-order ReaxFF to simulate the chemical reaction and study the damage to a large fragment of DNA-molecule in the solution by ionizing radiation. We illustrate that the randomly distributed clusters of diatomic OH-radicals that are primary products of megavoltage ionizing radiation in water-based systems are the main source of hydrogen-abstraction as well as formation of carbonyl- and hydroxyl-groups in the sugar-moiety that create holes in the sugar-rings. These holes grow up slowly between DNA-bases and DNA-backbone and the damage collectively propagate to DNA single and double strand break.

Abolfath, Ramin M; Brabec, Thomas

2011-01-01

257

Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis.  

PubMed

Successful DNA extraction is indispensable for molecular methods based on polymerase chain reaction (PCR); however, goat sperm DNA extraction is limited. Thus, the aim of this study was to evaluate three methods to extract DNA from goat sperm for use in PCR. Eight goat semen pools were used for DNA extraction by using the DNeasy Blood & Tissue Kit, phenol-chloroform, and Chelex-100 methods. DNA samples were analyzed spectrophotometrically to determine the DNA concentration and purity, visualized on 0.8% agarose gel, and used at different amounts (150, 100, 50, 10, and 1 ng) for PCR with electrophoresis, followed by 1.5% agarose gel electrophoresis. The quantity of DNA extracted with Chelex-100 was higher (P < 0.05) than that obtained with either the DNeasy Blood & Tissue Kit or the phenol-chloroform method, with the phenol-chloroform method yielding a greater quantity (P < 0.05) than the kit. The DNeasy Blood & Tissue Kit produced a higher (P < 0.05) purity product than the Chelex-100 method, and all samples obtained by the three protocols were positive for DNA, as assessed by electrophoresis. All of the different concentrations of DNA produced by these methods were amplified by PCR, although for DNA produced by the phenol-chloroform method, PCR was only possible after complementary purification. In conclusion, the Chelex-100 method is cheap, secure, simple, fast, and effective, and is a potential tool for extracting goat sperm DNA without limitations in PCR. PMID:25117364

Silva, E C B; Pelinca, M A; Acosta, A C; Silva, D M F; Gomes Filho, M A; Guerra, M M P

2014-01-01

258

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis  

PubMed Central

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65C2C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.2510?2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

2013-01-01

259

DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay.  

PubMed

The genotoxic activities of complex mixtures of organic extracts from the urban air particles collected in various localities of the Czech Republic, which differed in the extent and sources of air pollution, were compared. For this purpose, PM2.5 particles were collected by high volume samplers in the most polluted area of the Czech Republic--Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting a low level of air pollution--Trebon--a small town in the non-industrial region of Southern Bohemia. To prepare extractable organic matter (EOM), PM2.5 particles were extracted by dichloromethane and c-PAHs contents in the EOMs were determined. As markers of genotoxic potential, DNA adduct levels and oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG, levels) induced by EOMs in an acellular assay of calf thymus DNA coupled with P-postlabeling (DNA adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct levels (17.20 adducts/10? nucleotides/m vs. 8.49 adducts/10? nucleotides/m) were induced by EOM from Ostrava-Bartovice (immediate proximity of heavy industry) compared with that from Ostrava-Poruba (mostly traffic emissions). Oxidative DNA damage induced by EOM from Ostrava-Bartovice was more than fourfold higher than damage induced by EOM from Trebon (8-oxodG/10? dG/m: 0.131 vs. 0.030 for Ostrava-Bartovice vs. Trebon, respectively). Since PM2.5 particles collected in various localities differ with respect to their c-PAHs content, and c-PAHs significantly contribute to genotoxicity (DNA adduct levels), we suggest that monitoring of PM2.5 levels is not a sufficient basis to assess genotoxicity of respirable aerosols. It seems likely that the industrial emissions prevailing in Ostrava-Bartovice represent a substantially higher genotoxic risk than mostly traffic-related emissions in Ostrava-Poruba. B[a]P and c-PAH contents in EOMs are the most important factors relating to their genotoxic potential. PMID:21329747

Topinka, Jan; Rossner, Pavel; Milcova, Alena; Schmuczerova, Jana; Svecova, Vlasta; Sram, Radim J

2011-05-10

260

Promyelocytic leukemia nuclear bodies support a late step in DNA double-strand break repair by homologous recombination  

PubMed Central

SUMMARY The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RAR? fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2 fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20 fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RAR? fusion proteins disrupted PML-NB structure and reduced HRR by up to 10 fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia. PMID:22213200

Yeung, Percy Luk; Denissova, Natalia G.; Nasello, Cara; Hakhverdyan, Zhanna; Chen, J. Don; Brenneman, Mark A.

2012-01-01

261

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 Maxwell16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

2012-01-01

262

Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides.  

PubMed

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen. PMID:15543980

Shimelis, Olga; Zhou, Xiaojuan; Li, Guodong; Giese, Roger W

2004-10-22

263

Volume reduction solid phase extraction of DNA from dilute, large-volume biological samples.  

PubMed

Microdevices are often designed to process sample volumes on the order of tens of microliters and cannot typically accommodate larger volume samples without adversely affecting efficiency and greatly increasing analysis time. However, dilute, large-volume biological samples are frequently encountered, especially in forensic or clinical laboratories. A microdevice, capable of efficiently processing 0.5-1 mL samples has been developed for solid phase extraction (SPE) of DNA. SPE was carried out on a microdevice utilizing magnetic silica particles and an optimized volumetric flow rate and elution buffer, resulting in a 50-fold decrease in volume and a 15-fold increase in DNA concentration. Device characterization studies showed DNA extraction efficiencies comparable with previously reported silica-based purification methods, with robust performance demonstrated by the successful amplification of a fragment from the gelsolin gene extracted from dilute whole blood. In addition, the microchip-based method for SPE of large volume, dilute samples was also used to demonstrate the first successful on-chip purification of mitochondrial DNA (mtDNA) from both dilute whole blood and a degraded blood stain. PMID:20215033

Reedy, Carmen R; Bienvenue, Joan M; Coletta, Lisa; Strachan, Briony C; Bhatri, Naila; Greenspoon, Susan; Landers, James P

2010-04-01

264

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

Microsoft Academic Search

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

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

1998-01-01

265

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells  

Microsoft Academic Search

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared

C. N. M. Ribeiro; L. C. Peres; J. M. Pina-Neto

2004-01-01

266

A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.  

PubMed

Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. PMID:24631626

Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andrasson, Claes

2014-06-01

267

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

USGS Publications Warehouse

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

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

2009-01-01

268

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. PMID:21818364

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

269

Crude extracts of hepatoprotective plants, Solanum nigrum and Cichorium intybus inhibit free radical-mediated DNA damage  

Microsoft Academic Search

The presence of plant extracts of Solanum nigrum and Cichorium intybus in the reaction mixture containing calf thymus DNA and free radical generating system protect DNA against oxidative damage to its deoxyribose sugar moiety. The effect was dependent on the concentration of plant extracts. However, the effect of Cichorium intybus was much pronounced as compared to the effect of Solanum

Sarwat Sultana; Shahid Perwaiz; Mohammad Iqbal; Mohammad Athar

1995-01-01

270

Comparison of eight methods for the extraction of Bacillus atrophaeus spore DNA from eleven common interferents and a common swab.  

PubMed

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. PMID:21818364

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

271

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

PubMed Central

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

Fatima, Faria

2014-01-01

272

Polarized gonio fluorimetric measurements of DNA-chip: a step forward towards fluorescence quantification  

NASA Astrophysics Data System (ADS)

Quantifying hybridization and therefore fluorescence signals has become a key-issue in DNA-chip technology. Thus a better understanding of fluorescence near a surface has become a necessity. To study this issue, we modeled the fluorophore after an electromagnetic dipole radiating over the substrate; we then developed a simulation code which enabled us to calculate the observation-angle-dependent-intensity radiated by a population and altitude. In the mean time we developed a polarized-gonio-fluoimeter which permits angular fluorescence patterns and fluorescence polarization measurements. We studied DNA-chips obtained by covalent grafting of labeled oligonucleotides. Simulation curves perfectly matched experimental ones, enabling an accurate determination of fluorophore localization on the substrate. Once achieved a better understanding of the fluorophore emission, we designed and realized a thin-film-coated microscope slide dedicated to the enhancement of DNA-chip fluorescence. This substrate was used in a c-DNA gene analysis. Fluorescence enhancement was clearly observed enabling the detection of Cart1 and P2A which are undetectable when using non-coated microscope slides.

Barritault, Pierre; Getin, Stephane; Chaton, Patrick; Vinet, Francoise; Fouque, Brigitte; Stehle, Jean-Louis P.

2002-06-01

273

Cisplatin induced damage in kidney genomic DNA and nephrotoxicity in male rats: The protective effect of grape seed proanthocyanidin extract  

Microsoft Academic Search

The clinical use of cisplatin is highly limited, because of its renal toxicity. In this study, the protective effect of grape seed proanthocyanidin extract (GSPE) against cisplatin-induced nephrotoxicity is investigated in rats. Results showed that DNA qualitative analysis indicated an increase in the instability of the DNA purified from the cisplatin exposed kidney cells. Agarose gel electrophoresis revealed DNA damage

Abir A. Saad; Mokhtar I. Youssef; Lamiaa K. El-Shennawy

2009-01-01

274

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

E-print Network

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

275

Replication of Colicin E1 Plasmid DNA in Cell Extracts: II. Selective Synthesis of Early Replicative Intermediates*  

PubMed Central

The major products of colicin E1 plasmid DNA synthesis in cell extracts are completely replicated molecules and a class of molecules containing newly synthesized small DNA fragments. The addition of 10% glycerol and/or 2 mM spermidine to extracts blocks synthesis of completely replicated molecules while enhancing synthesis of molecules containing newly synthesized DNA fragments. The latter molecules, which contain on the average two DNA fragments of approximately 6 S, are early replicative intermediates for synthesis of completely replicated molecules. Synthesis of the intermediates is sensitive to rifampicin and depends on RNA synthesis. RNA components are linked to the 6S DNA molecules. PMID:4598305

Sakakibara, Yoshimasa; Tomizawa, Jun-Ichi

1974-01-01

276

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 3050 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m2 g?1) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)6, (dT)20, and (dT)40 DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules. PMID:22195031

Razaq, Aamir; Nystrm, Gustav; Strmme, Maria; Mihranyan, Albert; Nyholm, Leif

2011-01-01

277

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; Nystrm, Gustav; Strmme, Maria; Mihranyan, Albert; Nyholm, Leif

2011-01-01

278

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. PMID:20008460

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

2011-01-01

279

DNA extraction method using a silica-base resin type kit for the detection of genetically modified papaya.  

PubMed

Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya. PMID:18503240

Ohmori, Kiyomi; Tsuchiya, Hisayo; Watanabe, Takahiro; Akiyama, Hiroshi; Maitani, Tamio; Yamada, Toshiharu; Hirayama, Kuni; Satoh, Shuji

2008-04-01

280

DNA damage induced by wood preserving waste extracts in vitro without metabolic activation, as assayed by 32P-postlabeling.  

PubMed

Aqueous wood preserving waste (WPW) extracts were tested for their ability to damage DNA in vitro without metabolic activation. Two extracts were prepared from a surface tar and a surface clay soil sample of a WPW site. As assayed by 32P-post-labelling incubation of DNA with these extracts gave rise to highly complex, extract-specific profiles of DNA adducts whose formation depended on the concentration of WPW material. Most of the adducts appeared to be derived from polycyclic aromatic hydrocarbons (PAHs). Three mg organic WPW residue gave rise to total adduct levels of 13.8 (extract 1) and 66.2 (extract 2) DNA modifications in 10(7) DNA nucleotides, corresponding to 13.9 and 26.9 modifications, respectively, per 10 mg of soil. Thus, extract 2 was more active, although the parent residue had a 1.4-times lower PAH content as determined by gas chromatography/mass spectrometry (GC/MS). DNA adduct formation presumably was a consequence of (i) free radical reactions, possibly involving semiquinones and oxygen free radicals, and (ii) reaction of direct-acting electrophiles, derived from metabolism of WPW toxicants by soil microorganisms. These reactions appeared to be more active in sample 2. The results suggest that ground water at WPW sites contains DNA-reactive compounds posing a cancer hazard to humans. The in vitro DNA adduct assay represents a novel tool to readily assess this type of hazard and the possible effects of remediation measures. PMID:8062204

Randerath, K; Zhou, G D; Donnelly, K C; Safe, S H; Randerath, E

1994-08-15

281

A Two-Step Double Filter Method to Extract Open Water Surfaces from Landsat ETM+ Imagery  

NASA Astrophysics Data System (ADS)

In arid and semi-arid areas, lakes and temporal ponds play a significant role in agriculture and livelihood of local communities as well as in ecology. Monitoring the changes of these open water bodies allows to draw conclusions on water use as well as climatic impacts and can assist in the formulation of a sustainable resource management strategy. The simultaneous monitoring of larger numbers of water bodies with respect to their stage and area is feasible with the aid of remote sensing. Here the monitoring of lake surface areas is discussed. Landsat TM and ETM+ images provide a medium resolution of 30m, and offer an easily available data source to monitor the long term changes of water surfaces in arid and semi-arid regions. In the past great effort was put into developing simple indices to extract water surfaces from satellite images. However, there is a common problem in achieving accurate results with these indices: How to select a threshold value for water pixels without introducing excessive subjective judgment. The threshold value would also have to vary with location, land features and seasons, allowing for inherent uncertainty. A new method was developed using Landsat ETM+ imaginary (30 meter resolution) to extract open water surfaces. This method uses the Normalized Difference of Vegetation Index (NDVI) as the basis for an objective way of selecting threshold values of Modified Normalized Difference of Water Index (MNDWI) and Stress Degree Days (SDD), which were used as a combined filter to extract open water surfaces. We choose two study areas to verify the method. One study area is in Northeast China, where bigger lakes, smaller muddy ponds and wetlands are interspersed with agricultural land and salt crusts. The other one is Kafue Flats in Zambia, where seasonal floods of the Zambezi River create seasonal wetlands in addition to the more permanent water ponds and river channels. For both sites digital globe images of 0.5 meter resolution are available, which were taken within a few days of Landsat passing dates and which will serve here as ground truth information. On their basis the new method was compared to other available methods for extracting water pixels. Compared to the other methods, the new method can extract water surface not only from deep lakes/reservoirs and wetlands but also from small mud ponds in alkali flats and irrigation ponds in the fields. For the big and deep lakes, the extracted boundary of the lakes fits accurately the observed boundary. Five test sites in the study area in Northeast China with only shallow water surfaces were chosen and tested. The extracted water surfaces were compared with each site's digital globe maps, respectively to determine the accuracy of the method. The comparison shows that the method could extract all completely wet pixels (water area covering 100% of the pixel area) in all test sites. For partially wet pixels (50-100% of pixel area), the model can detect 91% of all pixels. No dry pixels were mistaken by the model as water pixels. Keywords: Remote sensing, Landsat ETM+ imaginary, Water Surface, NDVI, MNDWI, and SDD

Wang, Haijing; Kinzelbach, Wolfgang

2010-05-01

282

CDSbank: taxonomy-aware extraction, selection, renaming and formatting of protein-coding DNA or amino acid sequences  

PubMed Central

Background Protein-coding DNA sequences and their corresponding amino acid sequences are routinely used to study relationships between sequence, structure, function, and evolution. The rapidly growing size of sequence databases increases the power of such comparative analyses but it makes it more challenging to prepare high quality sequence data sets with control over redundancy, quality, completeness, formatting, and labeling. Software tools for some individual steps in this process exist but manual intervention remains a common and time consuming necessity. Description CDSbank is a database that stores both the protein-coding DNA sequence (CDS) and amino acid sequence for each protein annotated in Genbank. CDSbank also stores Genbank feature annotation, a flag to indicate incomplete 5? and 3? ends, full taxonomic data, and a heuristic to rank the scientific interest of each species. This rich information allows fully automated data set preparation with a level of sophistication that aims to meet or exceed manual processing. Defaults ensure ease of use for typical scenarios while allowing great flexibility when needed. Access is via a free web server at http://hazeslab.med.ualberta.ca/CDSbank/. Conclusions CDSbank presents a user-friendly web server to download, filter, format, and name large sequence data sets. Common usage scenarios can be accessed via pre-programmed default choices, while optional sections give full control over the processing pipeline. Particular strengths are: extract protein-coding DNA sequences just as easily as amino acid sequences, full access to taxonomy for labeling and filtering, awareness of incomplete sequences, and the ability to take one protein sequence and extract all synonymous CDS or identical protein sequences in other species. Finally, CDSbank can also create labeled property files to, for instance, annotate or re-label phylogenetic trees. PMID:24580755

2014-01-01

283

Extracting coordinated patterns of DNA methylation and gene expression in ovarian cancer  

PubMed Central

Objective DNA methylation, a regulator of gene expression, plays an important role in diverse biological processes including developmental process, carcinogenesis and aging. In particular, aberrant DNA methylation has been largely observed in several types of cancers. Currently, it is important to extract disease-specific gene sets associated with the regulation of DNA methylation. Materials and methods Here we propose a novel approach to find the minimum regulatory units of genes, co-methylated and co-expressed gene pairs (MEGP) that are highly correlated gene pairs between DNA methylation and gene expression showing the co-regulatory relationship. To evaluate whether our method is applicable to extract disease-associated genes, we applied our method to a large-scale dataset from the Cancer Genome Atlas extracting significantly associated MEGP and analyzed their functional correlation. Results We observed that many MEGP physically interacted with each other and showed high semantic similarity with gene ontology terms. Furthermore, we performed gene set enrichment tests to identify how they are correlated in a complex biological process. Our MEGP were highly enriched in the biological pathway associated with ovarian cancers. Conclusions Our approach is useful for discovering coordinated epigenetic markers associated with specific diseases. PMID:23599224

Joung, Je-Gun; Kim, Dokyoon; Kim, Kyung Hwa; Kim, Ju Han

2013-01-01

284

Antioxidant Activity and Protection from DNA Damage by Water Extract from Pine (Pinus densiflora) Bark  

PubMed Central

Water extract from Pinus densiflora (WPD) was investigated for its antioxidant activity and its ability to provide protection from DNA damage. A series of antioxidant assays, including a 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay, a reducing power assay, a metal-chelating assay, a superoxide radical scavenging assay, and a nitrite scavenging ability, as well as a DNA damage protection assay were performed. Total phenolic content was found to be 211.32 mg Tan/g WPD. The extract scavenged 50% DPPH free radical at a concentration of 21.35 ?g/mL. At that same concentration, the reducing power ability of WPD was higher than that of ?-tocopherol. The extract chelated 68.9% ferrous ion at the concentration of 4 mg/mL. WPD showed better nitrite scavenging effect at the lower pH. Meanwhile, WPD exhibited a strong capability for DNA damage protection at 1 mg/mL concentration. Taken together, these data suggest water extract from Pinus densiflora could be used as a suitable natural antioxidant. PMID:24471072

Jiang, Yunyao; Han, Woong; Shen, Ting; Wang, Myeong-Hyeon

2012-01-01

285

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; Sjberg, Per J R

2013-10-15

286

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis.  

PubMed

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA. PMID:17406572

Zhu, Kaichun; Jin, Huali; He, Zhonghuai; Zhu, Qinghong; Wang, Bin

2006-01-01

287

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; Pter Varnai

2005-01-01

288

Extraction of DNA from mucilaginous tissues of a sea slug (Elysia chlorotica).  

PubMed

Efforts to study the cellular and molecular biology of the symbiotic association between opisthobranch molluscs and algal chloroplasts have been hampered by the copious amounts of mucus produced by the animals. We report for the first time a procedure for isolating total DNA free of contaminating mucilaginous compounds from the mollusc Elysia chlorotica Gould that harbors photosynthetically active chloroplasts from the siphonaceous alga, Vaucheria litorea C. Agardh. This method involves an initial extraction of fresh or freeze-dried Elysia tissue in absolute ethanol and differential processing of the resultant two-phase pellet. Final purification by CsCl-gradient centrifugation produces high molecular weight DNA suitable for molecular analysis. PMID:7873179

Rumpho, M E; Mujer, C V; Andrews, D L; Manhart, J R; Pierce, S K

1994-12-01

289

Comparative evaluation of three different extraction methods for rice (Oryza sativa L.) genomic DNA.  

PubMed

Recently, more immediate and precise cultivar-identifying methods targeting the specific and/or introduced gene(s) have been put into practical use for various rice cultivars. However trustworthy and innovative the biotechnological analyses may be, DNA purity and quality do have unpredictable influences upon the identification. Extraction methods of rice DNA have hardly ever been compared in a comprehensive manner. In this study, we investigated extraction characteristics of three methods by using 10 rice cultivars and then examined template availability of rice DNAs thereby extracted. An UV spectrophotometric study with a view toward methods revealed three different facts: The Isoplant II kit method with inhibitor absorption yielded the most DNAs, the Takara kit method with magnetic trapping produced the best DNAs free from contaminative proteins, and the enzymatic digestion method exclusively with enzymatic digestions prepared the best DNAs free from contaminative polysaccharides. Moreover, with a view toward cultivars, an insignificant difference in yield was not entirely bore out, and some difference in cultivar might cause significant difference in yield; however, no significant difference in purity was found among the cultivars used. On the other hand, electrophoretic images of the DNAs from the same cultivars showed considerable differences in quality among the methods. Furthermore, the DNA extracts from certain brands of rice proved really available for cultivar identification by using polymerase chain reaction (PCR) related to sequence-tagged sites. Therefore, this study suggested that these extraction methods may be used as the situation demands and that the DNAs thereby extracted might work successfully even in cultivar-identifying PCRs. PMID:19253951

Sagi, Naoki; Monma, Kimio; Ibe, Akihiro; Kamata, Kunihiro

2009-04-01

290

An efficient protocol for total DNA extraction from the members of order Zingiberales- suitable for diverse PCR based downstream applications.  

PubMed

Different protocols are usually used for extracting total deoxyribonucleic acid (DNA) from different plant species of same order and DNA of the associated viruses. Here, we describe a rapid, efficient and universal protocol for isolating total DNA from the members of Zingiberales which harbor a high amount of polysaccharides and secondary metabolites. DNA isolated with this protocol was successfully used for PCR based downstream applications viz. random amplified polymorphic DNA (RAPD), Inter-simple sequence repeats (ISSR), DNA barcoding gene (Internal transcribed spacer and trnl-f) amplification and detection of the viruses. PMID:24363983

Devi, Khumallambam Devala; Punyarani, Kshetrimayum; Singh, Nandeibam Samarjit; Devi, Huidrom Sunitibala

2013-01-01

291

Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication.  

PubMed

Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC50 values ranging between 0.2 and 0.5?g/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection. PMID:25172789

Heidary Navid, M; Laszczyk-Lauer, M N; Reichling, J; Schnitzler, P

2014-09-25

292

Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.  

PubMed

A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety. PMID:19244904

Snchez, Beatriz; Rodrguez, Mar; Casado, Eva M; Martn, Alberto; Crdoba, Juan J

2008-12-01

293

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.  

PubMed

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006. PMID:18304664

Fredlund, Elisabeth; Gidlund, Ann; Olsen, Monica; Brjesson, Thomas; Spliid, Niels Henrik Hytte; Simonsson, Magnus

2008-04-01

294

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

295

A novel molecular protocol for the rapid extraction of DNA from bryophytes and the utility of direct amplification of DNA from a single dwarf male  

Microsoft Academic Search

A novel protocol for the rapid extraction of bryophyte DNA is presented and tested on nine mosses and one liverwort. Amplification products and sequences of the rps4 gene were obtained for all the samples tested. Direct amplification and sequencing of DNA from a single dwarf male was found to be possible. By adding single dwarf males of Dicranum scoparium directly

Niklas Pedersen; Stephen J. Russell; Angela E. Newton; Stephen W. Ansell

2006-01-01

296

DNA topoisomerase II? controls replication origin cluster licensing and firing time in Xenopus egg extracts  

PubMed Central

Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase II? (topo II?), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo II? around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo II? depletion accelerated origin cluster activation, and topo II? add-back negated overinitiation. Therefore, topo II? is not required for DNA replication, but topo II? clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo II? activity is dispensable for replication and revealing that topo II? clamps formed on replicating DNA do not block replication, presumably because topo II? acts behind and not in front of forks. Topo II? depletion increased, and topo II? addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo II? restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters. PMID:23757188

Gaggioli, Vincent; Le Viet, Barbara; Germe, Thomas; Hyrien, Olivier

2013-01-01

297

Novel DNA extraction assay for molecular identification of Aedes spp eggs.  

PubMed

Aedes aegypti and A. albopictus represent the two most important species of mosquitoes in relation to dengue virus transmission both in the Americas and Asia. However, the study of theses species generally requires the establishment of a colony for the larvae to hatch, or waiting for the adult development to perform its taxonomic classification, which is time consuming. Thus, the establishment of new methods aimed at obtaining DNA directly from the mosquito eggs is relevant. Accordingly, we compared a new approach based on Chelex() 100 resin with the standard STE method to extract DNA from the eggs of Aedes spp to molecularly identify these vectors. The Chelex() 100 resin approach was very efficient, as satisfactory amounts of DNA were obtained, making it possible to amplify and sequence a mitochondrial DNA barcode region widely used to identify species. The STE protocol yielded substantial amounts of DNA, but the 260/280 optical density ratio indicated a low quality, precluding amplification. This new method proved quite effective in obtaining DNA from even a single mosquito egg, and it can thus be applied in population genetic studies of various vector insects to enhance monitoring programs. PMID:25366769

Freitas, M T S; Gomes-Jnior, P P; Batista, M V A; Leal-Balbino, T C; Araujo, A L; Balbino, V Q

2014-01-01

298

Seabuckthron (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations.  

PubMed

In vitro assessment showed that H. rhamnoides (HrLE) extract possessed free radical scavenging activities and can protect gamma (gamma) radiation induced supercoiled DNA damage. For in vivo study, Swiss albino mice were administered with HrLE (30 mg/kg body weight) for 15 consecutive days before exposing them to a single dose of 5 Gy of beta radiation. HrLE significantly prevented the radiation induced genomic DNA damage indicated as a significant reduction in the comet parameters. The lipid peroxidation, liver function enzymes, expression of phosphorylated NFkappaB (p65) and IkappaBalpha increased whereas the endogenous antioxidants diminished upon radiation exposure compared to control. Pretreatment of HrLE extract ameliorated these changes. Based on the present results it can be concluded that H. rhamnoides possess a potential preventive element in planned and accidental nuclear exposures. PMID:25345244

Khan, Amitava; Manna, Krishnendu; Chinchubose; Das, Dipesh Kr; Sinha, Mahuya; Kesh, Swaraj Bandhu; Das, Ujjal; Dey, Rakhi Sharma; Banerji, Asoke; Dey, Sanjit

2014-10-01

299

DNA extraction Laboratory 1.03g, Donnan Laboratories, Crown Street, School of Biological Sciences, University of Liverpool. L69 7ZD.  

E-print Network

DNA extraction Laboratory 1.03g, Donnan Laboratories, Crown Street, School of Biological Sciences, University of Liverpool. L69 7ZD. Extraction of DNA from tissue: High salt method. Version 1.0, September://sciencepark.mdanderson.org/mbcore/protocols.html Any comments or suggestions should be sent to Phill Watts at p.c.watts@liv.ac.uk. #12;DNA extraction

Steve Kemp

300

Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR  

PubMed Central

Introduction: Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS). Materials and Methods: Sixteen venous blood samples collected in K3-EDTA tubes (400?l of whole blood) were used for the spotting (4 circles each 100?l) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of -thalassemia patients were also performed to ensure the quality of extraction. Results: The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ ?l was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/?l for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/?l when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA. Conclusion: It was concluded that the superparamagnetic-bead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes. PMID:24959449

2014-01-01

301

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

Microsoft Academic Search

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

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

2004-01-01

302

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies  

PubMed Central

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at ?20C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

Bali, Latifa El; Diman, Aurlie; Bernard, Alfred; Roosens, Nancy H. C.

2014-01-01

303

DNA extraction from arborescent monocots and how to deal with other challenging hosts.  

PubMed

Detection of pathogen DNA by polymerase chain reaction (PCR) assays is the most widely used method for diagnosing phytoplasma diseases. Reliable and efficient detection of phytoplasmas, especially in woody perennial plants, is challenging due to the unusually low abundance and sporadic distribution of phytoplasmas within infected host tissues. Detection success depends largely upon the host species and sampling procedures and, to a lesser extent, on the protocol used for DNA extraction. Here we describe a simple, straightforward, nondestructive stem sampling protocol to confirm phytoplasma infection of palms and other arborescent monocots of large stature. The protocol requires minimal processing of excised tissues and yields phytoplasma DNA preparations in suitable quantity for reliable detection by nested PCR assays. PMID:22987413

Harrison, Nigel A; Davis, Robert E; Helmick, Ericka E

2013-01-01

304

On-chip fraction collection for multiple selected ssDNA fragments using isolated extraction channels.  

PubMed

High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay. PMID:21227429

Li, Zheyu; Sun, Kai; Sunayama, Misato; Matsuo, Yasutaka; Mizeikis, Vygantas; Araki, Ryoko; Ueno, Kosei; Abe, Masumi; Misawa, Hiroaki

2011-02-18

305

Four DNA extraction methods used in loop-mediated isothermal amplification for rapid adenovirus detection.  

PubMed

Loop-mediated isothermal amplification (LAMP) assays have become powerful tools for rapid diagnosis of infectious diseases. A more efficient, convenient and cheaper method for template preparation from the pellets or supernatants of nasopharyngeal aspirates was sought. Three DNA extraction methods (boiling, boiling in 1% Triton X-100, and treating with 0.02M NaOH) were compared with the commonly used DNAzol DNA extraction method. DNA preparations were then subjected to adenovirus (ADV) detection using LAMP assays and 119 clinical samples. The specificities for all three methods were 100% compared with the DNAzol method. The sensitivity of the boiling method was greater than that for the other two methods. The templates extracted from supernatants of nasopharyngeal aspirates using the boiling technique were further evaluated. Higher sensitivity (90.9%) and specificity (96.5%) were observed for LAMP assays compared with those from quantitative PCR assays. In conclusion, for template preparation, boiling supernatants of nasopharyngeal aspirates had comparable sensitivity and specificity with the DNAzol method. There were the added advantages that the boiling technique was simpler, cheaper, and had a shorter processing time. The boiling technique could become a suitable substitute for the DNAzol method when LAMP assays are used for ADV detection. PMID:24747588

Sun, Yu; Zhao, Linqing; Zhao, Meng; Zhu, Runan; Deng, Jie; Wang, Fang; Li, Fan; Ding, Yaxin; Tian, Run; Qian, Yuan

2014-08-01

306

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; Prez, J M; Zhu, X Q

2009-03-01

307

Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.  

PubMed

Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing. PMID:24774752

Vo, A-T E; Jedlicka, J A

2014-11-01

308

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods.  

PubMed

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR. PMID:15537304

Peano, Clelia; Samson, Maria Cristina; Palmieri, Luisa; Gulli, Mariolina; Marmiroli, Nelson

2004-11-17

309

Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction.  

PubMed

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils. PMID:22493524

Hasan, Saba; Prakash, Jyoti; Vashishtha, Abhinav; Sharma, Agnivesh; Srivastava, Kuldeep; Sagar, Faizuddin; Khan, Nausheen; Dwivedi, Keshav; Jain, Payal; Shukla, Saransh; Gupta, Swati Prakash; Mishra, Saumya

2012-01-01

310

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.  

PubMed

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp circulating nucleic acid (CNA) kit, NucleoSpin Plasma XS (NS) kit and FitAmp plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. PMID:24853859

Devonshire, Alison S; Whale, Alexandra S; Gutteridge, Alice; Jones, Gerwyn; Cowen, Simon; Foy, Carole A; Huggett, Jim F

2014-10-01

311

RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps  

PubMed Central

The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein. PMID:24371286

Ronayne, Erin A.; Cox, Michael M.

2014-01-01

312

RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps.  

PubMed

The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein. PMID:24371286

Ronayne, Erin A; Cox, Michael M

2014-04-01

313

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. PMID:20392915

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

2010-01-01

314

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...

315

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 70C. 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; Gonzlez, P; Lorenzo, R A; Carro, A M

2011-09-28

316

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics  

PubMed Central

Under a tension of ?65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the ?-phage DNA with different melting degrees and temperatures (1025C). The shortening transient following a 2035 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 235 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

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

2014-01-01

317

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.  

PubMed

Under a tension of ?65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the ?-phage DNA with different melting degrees and temperatures (10-25C). The shortening transient following a 20-35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2-35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

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

2014-03-01

318

En masse retraction versus two-step retraction of anterior teeth in extraction treatment of bimaxillary protrusion.  

PubMed

In the present report, two techniques of space closure; two-step anterior teeth retraction (TSR) and en masse retraction (ER) were used in two adult patients who had bimaxillary protrusion and were treated with four premolar extractions and fixed orthodontic appliance therapy. Both patients had a Class I dental malocclusion and the same chief complaint, which is protrusive lips. Anterior teeth were retracted by two-step retraction; canine sliding followed by retraction of incisors with T-loop archwire in the first patient and by en masse retraction using Beta titanium alloy T-loop archwire in the second case. At the end of treatment, good balance and harmony of lips was achieved with maintenance of Class I relationships. The outcome of treatment was similar in the two patients with similar anchorage control. ER can be an acceptable alternative to the TSR during space closure since it is esthetically more acceptable. However, it requires accurate bending and positioning of the T-loop. PMID:24987640

Felemban, Nayef H; Al-Sulaimani, Fahad F; Murshid, Zuhair A; Hassan, Ali H

2013-01-01

319

En masse retraction versus two-step retraction of anterior teeth in extraction treatment of bimaxillary protrusion  

PubMed Central

In the present report, two techniques of space closure; two-step anterior teeth retraction (TSR) and en masse retraction (ER) were used in two adult patients who had bimaxillary protrusion and were treated with four premolar extractions and fixed orthodontic appliance therapy. Both patients had a Class I dental malocclusion and the same chief complaint, which is protrusive lips. Anterior teeth were retracted by two-step retraction; canine sliding followed by retraction of incisors with T-loop archwire in the first patient and by en masse retraction using Beta titanium alloy T-loop archwire in the second case. At the end of treatment, good balance and harmony of lips was achieved with maintenance of Class I relationships. The outcome of treatment was similar in the two patients with similar anchorage control. ER can be an acceptable alternative to the TSR during space closure since it is esthetically more acceptable. However, it requires accurate bending and positioning of the T-loop. PMID:24987640

Felemban, Nayef H.; Al-Sulaimani, Fahad F.; Murshid, Zuhair A.; Hassan, Ali H.

2013-01-01

320

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 234.84.8mgkg(-1), in the leaves of Betula pendula it was 107.41.8mgkg(-1) and in the plants of Artemisia vulgaris (root) and Medicago sativa (leaves) it amounted to 464.710.7mg kg(-1)and 146.81.2mgkg(-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

2013-09-30

321

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.  

PubMed

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit. PMID:24047134

Bai, Jinhe; Baldwin, Elizabeth; Liao, Hui-Ling; Zhao, Wei; Kostenyuk, Igor; Burns, Jacqueline; Irey, Mike

2013-10-01

322

The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.  

ERIC Educational Resources Information Center

Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

Falconer, A. C.; Hayes, L. J.

1986-01-01

323

A one-step extraction procedure for the screening of cocaine, amphetamines and cannabinoids in postmortem blood samples.  

PubMed

A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification in postmortem whole blood samples of cocaine (COC), amphetamines (AMPs) and cannabis; the main drugs involved in cases of impaired driving in Brazil. The analytes were extracted by solid-phase extraction by means of Bond-Elute Certify cartridges, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide at 80C for 30 min and analyzed by GC-MS. Linearity ranged from 10 to 500 ng/mL, except for ecgonine methyl ester, for which linearity ranged from 10 to 100 ng/mL. Inter- and intra-day imprecision ranged from 2.8 to 18.4% and from 1.5 to 14.9%, respectively. Accuracy values lay between 86.9 and 104.4%. The limit of quantitation for all drugs was 10 ng/mL and recoveries were >74% for all analytes, except for cannabinoids, which showed poor recovery (?30%). The developed method was applied to real samples collected from deceased victims due to traffic accidents. These samples were selected according to the results obtained in immunoassay screening on collected urine samples. Five samples were positive for the presence of COC and metabolites, four samples were positive for cannabinoids, six samples were positive for AMPs and two samples were drug negative. Some samples were positive for more than one class of drug. Results obtained from whole blood samples showed good agreement with urine screening. The developed method proved capable of quantifying all three classes of drugs of abuse proposed in this study, through a one-step extraction procedure. PMID:24782143

Pelio, Fabrcio Souza; Peres, Mariana Dadalto; Pissinate, Jauber Fornaciari; De Martinis, Bruno Spinosa

2014-01-01

324

Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences  

PubMed Central

Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements. PMID:18830455

Lampronti, Ilaria; Khan, Mahmud T.H.; Borgatti, Monica; Bianchi, Nicoletta

2008-01-01

325

Evaluation of five protocols for DNA extraction from leaves of Malus sieversii, Vitis vinifera, and Armeniaca vulgaris.  

PubMed

Leaves of Malus sieversii, Vitis vinifera, and Armeniaca vulgaris contain substantial amounts of secondary metabolites, which limit the high-quality DNA extraction performance. In this study, five extraction protocols were compared for their ability to produce good quality DNA from fresh and dried (with silica gel) leaves of these species. The modified protocol of Dellaporta et al., using polyvinylpyrrolidone to bind the phenolic compounds and a high molar concentration of potassium acetate to inhibit co-precipitation of polysaccharides with DNA, produced the best DNA quality for all species tested. DNA extracted by this method had a 1.77-1.96 A260/280 nm ratio and successful amplification of the 18S ribosomal DNA gene. DNA concentrations of dried leaves were lower than those obtained from fresh leaves, which was likely due to aspects of the drying procedure. All five methods for grapevine produced DNA of obvious better quality from green canes compared to leaves, due to the relatively low content of secondary metabolites in the former. For grapevine and apricot, three methods can be equally used to obtain DNA of good quality: the Doyle and Doyle modified method using CTAB and high concentration of NaCl, the Jobes et al. modified method, and the sodium dodecyl sulfate mini preparation method of Edwards et al. The protocol of Jobes et al. using LiCl for RNA removal showed the best results for most of the M. sieversii samples examined. PMID:24634185

Aubakirova, K; Omasheva, M; Ryabushkina, N; Tazhibaev, T; Kampitova, G; Galiakparov, N

2014-01-01

326

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

PubMed

Bloodstains were made with 200 microliters 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 given category. With a DNA yield of up to 10 micrograms, paper, glass, nylon, wood, smooth leather and wool gave the best results, followed by blue denim and wallpaper (up to 6 micrograms), cotton fabric and carpeting (up to 4 micrograms) and suede (up to 2 micrograms). For several stain carriers the DNA-containing solution was contaminated by chemical substances, which in the case of the blue denim, suede, and carpet samples inhibited the digestion of the DNA with restriction enzymes and prevented DNA typing. The different textures of the stain carriers tested and (as for varying yields on the same carrier) the differing degree of loss of DNA during extraction and the physiological variation in the number of leukocytes in human blood are discussed as possible reasons for the wide range of variation in the amounts of DNA it was possible to extract. PMID:1968692

Prinz, M; Berghaus, G

1990-01-01

327

Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis  

Microsoft Academic Search

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 chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid

Richard A Haugland; John L Heckman; Larry J Wymer

1999-01-01

328

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

2009-01-01

329

Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.  

PubMed

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system. PMID:21694765

Sankaranarayanan, Krithivasan; Timofeeff, Michael N; Spathis, Rita; Lowenstein, Tim K; Lum, J Koji

2011-01-01

330

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. PMID:24089629

Ke, Yuebin; Xu, Xinyun; Wu, Shuang; Huang, Juan; Misra, Hara; Li, Yunbo

2013-01-01

331

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 H?O?-induced DNA damage. These protective effects may be related to its high total phenolic content (17.650.97 mg GAE/g), total flavonoid content (8.040.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.480.21 mg Na?EDTA/g). PMID:23871827

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

2013-09-01

332

Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction  

PubMed Central

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system. PMID:21694765

Sankaranarayanan, Krithivasan; Timofeeff, Michael N.; Spathis, Rita; Lowenstein, Tim K.; Lum, J. Koji

2011-01-01

333

5-Methylation of Cytosine in CG:CG Base-pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics  

PubMed Central

Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile opening mode and two shear sliding and tearing modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics. PMID:24313757

Yusufaly, Tahir I.; Li, Yun; Olson, Wilma K.

2014-01-01

334

Mismatch repair in Xenopus egg extracts: DNA strand breaks act as signals rather than excision points.  

PubMed Central

In Xenopus egg extracts, DNA strand breaks (nicks) located 3' or 5' to a mismatch cause an overall 3-fold stimulation of the repair of the mismatch in circular heteroduplex DNA molecules. The increase in mismatch repair is almost entirely due to an increase in repair of the nicked strand, which is stimulated 5-fold. Repair synthesis is centered to the mismatch site, decreases symmetrically on both sides, and its position is not significantly altered by the presence of the nick. Therefore, it appears that in the Xenopus germ cells, the mismatch repair system utilizes nicks as signals for the induction and direction of mismatch repair, but not as the start or end point for excision and resynthesis. Images Fig. 3 PMID:8816768

Varlet, I; Canard, B; Brooks, P; Cerovic, G; Radman, M

1996-01-01

335

DNA DNA DNA (d)DNA DNA DNA  

E-print Network

DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

Hagiya, Masami

336

A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva  

PubMed Central

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (812 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device. PMID:23370016

Oblath, Emily A.; Henley, W. Hampton; Alarie, Jean Pierre

2013-01-01

337

[DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].  

PubMed

The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes. PMID:20209349

Cardozo, Daniela Maira; Guelsin, Glucia Andria; Clementino, Samaia Laface; Melo, Fabiano Cavalcante de; Braga, Marco Antnio; Souza, Cleonice de; Moliterno, Ricardo Alberto; Visentainer, Jeane Eliete Laguila

2009-01-01

338

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 4.25105 cells g?1 using Griffiths and 4.25106 cells g?1 using FastDNA Spin kit. PMID:21448453

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

339

Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR  

PubMed Central

Objective The aim of this study was to evaluate, by PCR-RFLP and Real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. Material and Methods A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by DNA extraction. The concentration, purity and integrity of the DNA were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 A/G (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using Real-Time PCR. Results There was no significant difference of DNA yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and Real time PCR reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by Real-Time PCR presented C allele for IRF6 gene polymorphism (homozygous: CC; heterozygous: CT) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. Conclusion We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and PCR amplified after storage in mouthwash solution at room temperature. PMID:23032210

KUCHLER, Erika Calvano; TANNURE, Patricia Nivoloni; FALAGAN-LOTSCH, Priscila; LOPES, Taliria Silva; GRANJEIRO, Jose Mauro; AMORIM, Lidia Maria Fonte

2012-01-01

340

Pretreatment with Restriction Enzyme or Bovine Serum Albumin for Effective PCR Amplification of Epstein-Barr Virus DNA in DNA Extracted from Paraffin-Embedded Gastric Carcinoma Tissue  

Microsoft Academic Search

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced

YUKIO SATOH; NORIKO TAKASAKA; YOSHIKO HOSHIKAWA; MITSUHIKO OSAKI; SATOSHI OHFUJI; HISAO ITO; NOBUAKI KAIBARA; TAKESHI KURATA; TAKESHI SAIRENJI

1998-01-01

341

Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells  

SciTech Connect

In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

Roper, Katherine; Coverley, Dawn, E-mail: dc17@york.ac.uk

2012-03-10

342

Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR  

PubMed Central

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5104 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.

2013-01-01

343

Evaluation of two DNA extraction methods from maternal plasma for using in non-invasive bovine fetus gender determination  

PubMed Central

Background: Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8th week of pregnancy, by maternal blood sample testing. Objective: The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Materials and Methods: Maternal blood samples were taken from 40 pregnant cows during the 8th-38th weeks of gestation. DNA was extracted from 350 l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A260 and purity (A260/A280) of extracted DNA were detected by ultraviolet spectrophotometer. Three l of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. Results: The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 (p>0.05, p=0.3549), but the difference between mean purity (A260/A280) of DNA extracted by phenol-chloroform method and salting-out method was significant (p<0.001). X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. Conclusion: The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma.

Davoudi, Arash; Tarang, Alireza; Aleyasin, Seyed Ahmad; Salehi, Abdolreza; Seighalani, Ramin; Tahmoressi, Farideh

2012-01-01

344

Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis.  

PubMed

Tagetes, a genus of flowering marigolds in the family Asteraceae (Compositeae), is reported to be a medicinal plant with hypotensive, spasmolytic, anti-inflammatory, antimicrobial, and antifungal properties. Tagetes minuta characteristically contains high concentrations of essential oils, flavonoids, polyphenols, and polysaccharides that interfere with DNA, causing erroneous or no PCR products. We tested and modified various standard protocols in an effort to isolate high-quality DNA from different plant tissues of T. minuta. We used sun-dried, shade-dried and fresh-leaf tissues, as well as seeds for DNA analysis. The DNA obtained from seeds and fresh-leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. We were able to obtain good quality DNA from fresh leaf tissues without using liquid nitrogen. A relatively large amount of DNA was also extracted from the sun- and shade-dried tissues, but its quality was not as good as that from seeds. The DNA extracted from seeds and fresh leaves was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extracting high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils. PMID:20309824

Shahzadi, I; Ahmed, R; Hassan, A; Shah, M M

2010-01-01

345

A rapid in vitro polyomavirus DNA replication assay  

Microsoft Academic Search

Traditionally, the Hirt extraction method, a multi-step, labor-intensive and time-consuming procedure, is employed to extract selectively low- molecular weight DNA for polyomavirus DNA replication analyses. DNA replication results obtained with this approach are often inconsistent between replicate samples. To increase the efficiency and reproducibility of the polyomavirus DNA replication assay, we compared the DNA quality and yield using Qiagen Spin

Katja Ziegler; Thomas Bui; Richard J. Frisque; John A. Burns

346

A rapid in vitro polyomavirus DNA replication assay  

Microsoft Academic Search

Traditionally, the Hirt extraction method, a multi-step, labor-intensive and time-consuming procedure, is employed to extract selectively low-molecular weight DNA for polyomavirus DNA replication analyses. DNA replication results obtained with this approach are often inconsistent between replicate samples. To increase the efficiency and reproducibility of the polyomavirus DNA replication assay, we compared the DNA quality and yield using Qiagen Spin Column

Katja Ziegler; Thomas Bui; Richard J. Frisque; Andrew Grandinetti; Vivek. R. Nerurkar

2004-01-01

347

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

2014-02-15

348

Improved analytical procedure for determination of chlorinated pesticide residues in human serum using solid phase disc extraction (SPDE), single-step clean-up and gas chromatography  

Microsoft Academic Search

SummaryAn improved analytical methodology based on solid-phase disc extraction (SPDE) and a single-step clean-up on Florisil is proposed\\u000a for a large number of organochlorine pesticide residues in serum. Extraction was performed following denaturation of proteins\\u000a with formic acid after it was shown that it has no degradation effect on targeted analytes (?, ?, ?-HCH isomers, HCB, DDT\\u000a with its 5

P. Manirakiza; A. Covaci; P. Schepens

2002-01-01

349

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods  

NASA Astrophysics Data System (ADS)

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

2010-12-01

350

Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus.  

PubMed

Procedures for the purification of virions and nucleocapsids of Baculovirus penaei (BP) of penaeid shrimp and subsequent extraction of the viral nucleic acid are described. BP-infected hepatopancrata, from two species of shrimp from different geographical locations in the Americas, were removed and homogenized in a solution of TN buffer (0.01 M Tris-HCl, 0.10 M NaCl, pH 8.0). The homogenized mixture was strained through a 100-mesh screen to remove large pieces of tissue and centrifuged to concentrate the remaining material. The pellet was suspended in TN buffer and layered on to a handmade CsCl gradient. Fractions were collected according to the bands observed in the gradient, and the optical density at 254 nm was recorded for each fraction. The resultant data was tabulated and graphed. Additionally, each fraction was examined by transmission electron microscopy to determine relative numbers of viral particles present. Large amounts of virus consistently corresponded to a specific band in the gradient, which produced a peak when the spectrophometric data was graphed. Nucleic acid was then extracted from the purified viral particles. Removal of polysaccharides was accomplished with the addition of CTAB/NaCl. The BP DNA was visualized on an agarose gel with phage lambda DNA markers for size estimation, and a preliminary endonuclease digestion was performed using BamHI. PMID:1660489

Bruce, L D; Trumper, B B; Lightner, D V

1991-10-01

351

Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends.  

PubMed

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1. PMID:10793133

Guo, Z; Dunphy, W G

2000-05-01

352

Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy.  

PubMed

Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65C to 90C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy. PMID:22414380

Rickerts, Volker; Khot, Prasanna D; Ko, Daisy L; Fredricks, David N

2012-08-01

353

One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity.  

PubMed

An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed. Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli. Approx. 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S). h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step. From 1 l of cell culture, approx. 0.1 mg of purified h-TR beta 1-S was obtained. The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity. The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity. In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor. These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity. The purified h-TR beta 1-S is suitable for structural and functional studies. This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast. PMID:8227948

Park, J B; Ashizawa, K; Parkison, C; Cheng, S Y

1993-09-01

354

High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system.  

PubMed

The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated. PMID:16377204

Blommel, Paul G; Martin, Peter A; Wrobel, Russell L; Steffen, Eric; Fox, Brian G

2006-06-01

355

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

356

Novel DNA Extraction Method Unveiled the Ancient Hot Deep Biosphere Concealed in Terrestrial Sedimentary Rocks  

NASA Astrophysics Data System (ADS)

It has been proposed that the hot deep biosphere is distributed deeply in the crust of the Earth. Below the upper limit of life, prokaryotic habitats extend at a depth of 4 km within a typical range of thermal gradients (2.5-3C/100m). In contrary, large geothermal gradients allow the hot deep biosphere approaching to the Earths surface. We conducted aseptic and deoxygenated drilling targeting Miocene marine siliceous rocks in a tectonically stable inland fore-arc basin in central Japan. Although the in-situ groundwater temperature was 30.2C at a maximum drilling depth of 352 mbgl, opal-CT and clinoptilolite, which commonly form as a result of progressive burial at 30-60C and over 70C, respectively, were detected by XRD analysis. However, burial-driven degradation and transformation of sterols (sterene to sterane) was not evident in the core samples. As presented by Y. Suzuki et al. in this meeting, extant microbial populations were dominated by Pseudomonas spp. and Flavobacterium spp. with cell numbers ranging ~107-108 cells/cm3 rock. Despite the abundance of microbial cells, DNA were not extracted from the core samples by conventional methods. We developed a DNA extraction method to avoid binding of DNA onto the siliceous mineral matrix by heating under alkaline conditions, which resulted in the successful retrieval of PCR-amplifiable DNA. Unexpectedly, 16S rRNA gene sequences closely related thermophilic Geobacillus stearothermophilus and Thermus thermophilus were dominant in the clone libraries from 300- and 350-m deep core samples, while those almost identical to the Pseudomonas spp. were minor. Based on the correlation between the GC contents of 16S rRNA gene sequences and growth temperatures of prokaryotes, the estimated growth temperatures were 90.0C (G+C=66.3%) and 67.5C (G+C=61.3%) for G. stearothermophilus and T. thermophilus, respectively. From these results, it is implied that temperature rise in the past led to the colonization of thermophilic bacteria and the transformation of silica minerals in the deep subsurface. As intensive erosion is unlikely around the drilling site, a short period of hydrothermal activities rather than long-term burial at great depth caused high temperature conditions, which might explain the lack of maturity in hydrocarbon. A novel DNA-based approach coupled mineralogical and organic geochemical analyses has the potential to reconstruct ancient biogeochemical processes mediated in the deep subsurface as well as geothermal history. This study was supported by grants from the Nuclear and Industrial Safety Agency (NISA) and Japan Nuclear Energy Safety Organization (JNES).

Kouduka, M.; Suko, T.; Okuzawa, K.; Fukuda, A.; Nanba, K.; Yamamoto, M.; Sakata, S.; Ito, K.; Suzuki, Y.

2009-12-01

357

Molecular Dynamics Simulations of the 136 Unique Tetranucleotide Sequences of DNA Oligonucleotides. II: Sequence Context Effects on the Dynamical Structures of the 10 Unique Dinucleotide Steps  

PubMed Central

Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA. Calculations were carried out on the 136 cases embedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All simulations were carried out using a well-defined state-of-the-art MD protocol, the AMBER suite of programs, and the parm94 force field. In a previous article (Beveridge et al. 2004. Biophysical Journal. 87:37993813), the research design, details of the simulation protocol, and informatics issues were described. Preliminary results from 15 ns MD trajectories were presented for the d(CpG) step in all 10 unique sequence contexts. The results indicated the sequence context effects to be small for this step, but revealed that MD on DNA at this length of trajectory is subject to surprisingly persistent cooperative transitions of the sugar-phosphate backbone torsion angles ? and ?. In this article, we report detailed analysis of the entire trajectory database and occurrence of various conformational substates and its impact on studies of context effects. The analysis reveals a possible direct correspondence between the sequence-dependent dynamical tendencies of DNA structure and the tendency to undergo transitions that trap them in nonstandard conformational substates. The difference in mean of the observed basepair step helicoidal parameter distribution with different flanking sequence sometimes differs by as much as one standard deviation, indicating that the extent of sequence effects could be significant. The observations reveal that the impact of a flexible dinucleotide such as CpG could extend beyond the immediate basepair neighbors. The results in general provide new insight into MD on DNA and the sequence-dependent dynamical structural characteristics of DNA. PMID:16169978

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

2005-01-01

358

Interlaboratory Evaluation of Different Extraction and Real-Time PCR Methods for Detection of Coxiella burnetii DNA in Serum ?  

PubMed Central

In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever. PMID:20826645

Tilburg, Jeroen J. H. C.; Melchers, Willem J. G.; Pettersson, Annika M.; Rossen, John W. A.; Hermans, Mirjam H. A.; van Hannen, Erik J.; Nabuurs-Franssen, Marrigje H.; de Vries, Maaike C.; Horrevorts, Alphons M.; Klaassen, Corne H. W.

2010-01-01

359

Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis.  

PubMed

We evaluated the ability of 3 kits: QIAmp DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification, and PureLink Genomic DNA (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and ?-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for ?-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the ?-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale. PMID:24207076

Uda-Shimoda, Carla Fernanda; Colli, Cristiane Maria; Pavanelli, Mariana Felgueira; Falavigna-Guilherme, Ana Lcia; Gomes, Mnica Lcia

2014-01-01

360

Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.  

PubMed

Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery. PMID:18950887

Jara, C; Mateo, E; Guillamn, J M; Torija, M J; Mas, A

2008-12-10

361

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 0.51.0 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 0.51.0 g material and real-time PCR with SYBR Green fluorescence detection was developed. PMID:19330077

Brandfass, Christoph; Karlovsky, Petr

2008-01-01

362

Object extraction Object extraction  

E-print Network

Object extraction #12;Object extraction · Extracting topographic objects from images · the main goal of aerial photogrammetry · object extraction consists of two steps · image interpretation extraction · Extracting topographic objects from images · identify all objects of a certain class · measure

Giger, Christine

363

Sensing cocaine in saliva with attenuated total reflection infrared (ATR-IR) spectroscopy combined with a one-step extraction method  

NASA Astrophysics Data System (ADS)

On-site drug tests have gained importance, e.g., for protecting the society from impaired drivers. Since today's drug tests are majorly only positive/negative, there is a great need for a reliable, portable and preferentially quantitative drug test. In the project IrSens we aim to bridge this gap with the development of an optical sensor platform based on infrared spectroscopy and focus on cocaine detection in saliva. We combine a one-step extraction method, a sample drying technique and infrared attenuated total reflection (ATR) spectroscopy. As a first step we have developed an extraction technique that allows us to extract cocaine from saliva to an almost infrared-transparent solvent and to record ATR spectra with a commercially available Fourier Transform-infrared spectrometer. To the best of our knowledge this is the first time that such a simple and easy-to-use one-step extraction method is used to transfer cocaine from saliva into an organic solvent and detect it quantitatively. With this new method we are able to reach a current limit of detection around 10 ?g/ml. This new extraction method could also be applied to waste water monitoring and controlling caffeine content in beverages.

Hans, Kerstin M.-C.; Gianella, Michele; Sigrist, Markus W.

2012-03-01

364

Mild Two-Step Method to Construct DNA-Conjugated Silicon Nanoparticles: Scaffolds for the Detection of MicroRNA-21.  

PubMed

We describe a novel two-step method, starting from bulk silicon wafers, to construct DNA conjugated silicon nanoparticles (SiNPs). This method first utilizes reactive high-energy ball milling (RHEBM) to obtain alkene grafted SiNPs. The alkene moieties are subsequently reacted with commercially available thiol-functionalized DNA via thiol-ene click chemistry to produce SiNP DNA conjugates wherein the DNA is attached through a covalent thioether bond. Further, to show the utility of this synthetic strategy, we illustrate how these SiNP ODN conjugates can detect cancer-associated miR-21 via a fluorescence ON strategy. Given that an array of biological molecules can be prepared with thiol termini and that SiNPs are biocompatible and biodegradable, we envision that this synthetic protocol will find utility in salient SiNP systems for potential therapeutic and diagnostic applications. PMID:25243490

Su, Xiaoye; Kuang, Li; Battle, Cooper; Shaner, Ted; Mitchell, Brian S; Fink, Mark J; Jayawickramarajah, Janarthanan

2014-10-15

365

Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes  

PubMed Central

Background Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). Methods Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. Results The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. Conclusion Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants. PMID:24279749

2013-01-01

366

Evaluation of a semi-automated, magnetic bead-based DNA extraction method for genetic fingerprinting of forensic casework samples  

Microsoft Academic Search

In order to cope with the demanding workload for DNA profiling of forensic casework samples a concept for a semi-automated processing system was developed at the Landeskriminalamt (Office of Criminal Investigation) Baden-Wrttemberg, Germany [K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, this issue]. The applied magnetic bead extraction method is based

Barbara Haak; Andrea Porsche; Kai Vollack; Peter Zimmermann; Werner Pflug

2008-01-01

367

A rapid method for extraction of cotton ( Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis  

Microsoft Academic Search

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances.\\u000a We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP\\u000a and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol\\u000a oxidase inhibitors

Andrew H. Paterson; Curt L. Brubaker; Jonathan F. Wendel

1993-01-01

368

Comparison of small-scale methods for the rapid extraction of plant DNA suitable for PCR analysis  

Microsoft Academic Search

We present results from a comparison of six methods for rapid DNA extraction from leaf and other plant tissues. We have used\\u000a samples from six plant species in our study, including both crop species and their wild relatives. The success of the methods\\u000a is assessed by PCR of the DNA using conserved primers, and the applicability of the different methods

Hilary J. Rogers; Nigel A. Burns; Helen C. Parkes

1996-01-01

369

High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method.  

PubMed

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community. PMID:20118363

Urakawa, Hidetoshi; Martens-Habbena, Willm; Stahl, David A

2010-04-01

370

Critical points of DNA quantification by real-time PCR - effects of DNA extraction method and sample matrix on quantification of genetically modified organisms  

PubMed Central

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR. PMID:16907967

Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

2006-01-01

371

Ion-Channel Genosensor for the Detection of Specific DNA Sequences Derived from Plum Pox Virus in Plant Extracts  

PubMed Central

A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3?/4? as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 18 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 1050 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal. PMID:25302809

Malecka, Kamila; Michalczuk, Lech; Radecka, Hanna; Radecki, Jerzy

2014-01-01

372

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts  

PubMed Central

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5?-base overhang. Regardless of the 5?-end structure, all bleomycin-induced DSBs possess 3?-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of ?0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires ?100 ng substrate DNA and ?50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA. PMID:11504886

Pastwa, Elzbieta; Neumann, Ronald D.; Winters, Thomas A.

2001-01-01

373

Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting  

Microsoft Academic Search

We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns\\u000a obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods,\\u000a i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction\\u000a followed by DNA extraction, and

J. Kozdrj; J. D. van Elsas

2000-01-01

374

Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction  

PubMed Central

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results. PMID:12791844

Honore-Bouakline, S.; Vincensini, J. P.; Giacuzzo, V.; Lagrange, P. H.; Herrmann, J. L.

2003-01-01

375

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay.  

PubMed

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method. PMID:20458155

Hyeon, Ji Yeon; Hwang, In Gyun; Kwak, Hyo Sun; Park, Chankyu; Choi, In Soo; Seo, Kun Ho

2010-06-01

376

High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.  

PubMed

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively). PMID:23793016

Holmberg, Rebecca C; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G; Chandler, Darrell P

2013-01-01

377

Effects of the most common methods for the enhancement of latent fingerprints on DNA extraction from forensic samples  

Microsoft Academic Search

The aim of the research was to understand if the use of chemicals compounds used to enhance latent fingerprints, might interfere with the extraction and amplification of DNA from biological samples on crime scenes. Only three methods were used: powders (black and white ones, used on non porous surfaces, and here applied on glass), cyanoacrylate (used on non porous surfaces,

S. Gino; M. Omedei

378

Evaluation of DNA extraction methods from mouse stomachs for the quantification of H. pylori by real-time PCR  

Microsoft Academic Search

Real-time PCR methods have recently been developed for the quantification of Helicobacter pylori from infected mouse stomachs. However, the extent to which results is affected by the efficiency of different methods of DNA extraction and the degree of inhibition of the subsequent PCR have largely been ignored. In this study, mouse stomachs were processed using two homogenisation methods: complete disruption

Yvonne Roussel; Mark Wilks; Andrew Harris; Charles Mein; Soad Tabaqchali

2005-01-01

379

Phenolic composition, DNA damage protective activity and hepatoprotective effect of free phenolic extract from Sphallerocarpus gracilis seeds.  

PubMed

The phenolic composition of the free phenolic extract from Sphallerocarpus gracilis seeds was analyzed by HPLC-MS and predominant compounds were chlorogenic acid, di-caffeoylquinic acid glucoside and luteolin-7-O-glucoside. The free phenolic extract was evaluated for DNA damage protective activity induced by ROO and OH radicals and hepatoprotective effect in vivo and in vitro. Results revealed that the free phenolic extract exhibited significant protective activity against both ROO and OH radical-induced DNA damage and the phenolic extract exerted more potent inhibitory activity against OH radical-induced damage than against that induced by ROO radicals. In vivo experimental results showed that the phenolic extract significantly prevented the increase of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities and hepatic malondialdehyde level caused by CCl4 in rats, and markedly increased hepatic superoxide dismutase, catalase and glutathione peroxidase levels. Histopathological examinations further confirmed that the phenolic extract could protect the liver from CCl4-induced damage. In vitro experimental results showed that the phenolic extract could reduce BRL hepatocyte apoptosis and damage induced by CCl4. These findings indicate that the S. gracilis seed could be developed as a medicinal herb for the therapy and prevention of hepatic injury. PMID:24657314

Gao, Chun-yan; Tian, Cheng-rui; Zhou, Rui; Zhang, Run-guang; Lu, Yue-hong

2014-05-01

380

A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids  

Microsoft Academic Search

One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GCMS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs.

Sodeif Azadmard-Damirchi; Paresh C. Dutta

2009-01-01

381

Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: The inhibitory effect of linoleic acid on the DNA-binding step  

SciTech Connect

c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90({+-}0.54) x 10{sup -7} M for Max85/Max85 homodimer, 6.85({+-}0.25) x 10{sup -3} M for Myc87/Myc87 homodimer, and 2.55({+-}0.29) x 10{sup -8} M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33({+-}0.21) x 10{sup -9} M for Max85/Max85/DNA, 2.27({+-}0.08) x 10{sup -12} M for Myc87/Myc87/DNA, and 4.43({+-}0.37) x 10{sup -10} M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.

Jung, Kyung Chae [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Rhee, Ho Sung [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Park, Chi Hoon [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Yang, Chul-Hak [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of)]. E-mail: chulyang@plaza.snu.ac.kr

2005-08-19

382

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development  

EPA Science Inventory

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

383

Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods  

Microsoft Academic Search

Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains

C. Jara; E. Mateo; J. M. Guillamn; M. J. Torija; A. Mas

2008-01-01

384

Cisplatin induced damage in kidney genomic DNA and nephrotoxicity in male rats: the protective effect of grape seed proanthocyanidin extract.  

PubMed

The clinical use of cisplatin is highly limited, because of its renal toxicity. In this study, the protective effect of grape seed proanthocyanidin extract (GSPE) against cisplatin-induced nephrotoxicity is investigated in rats. Results showed that DNA qualitative analysis indicated an increase in the instability of the DNA purified from the cisplatin exposed kidney cells. Agarose gel electrophoresis revealed DNA damage in the form of smearing as well as ladder like fragmentation of the kidney genomic DNA. Cisplatin produced different RAPD patterns compared to control. Deletion of bands for the amplified DNA extracted from cisplatin treated rats was the most common outcome. Treatment with cisplatin decreased albumin, and increased urea and creatinine. Cisplatin significantly increased the level of kidney free radicals, and decreased the glutathione content and the activities of the antioxidant enzymes. The presence of GSPE with cisplatin significantly alleviated its nephrotoxicity. In conclusion, the present study showed that cisplatin induced damage in the kidney genomic DNA, lipid peroxidation, inhibition of antioxidant enzymes and alterations of biochemical parameters in plasma and kidney of rats. While, GSPE treatment protected against the toxic effects induced by cisplatin. Thus, GSPE may be used to prevent toxicity during chemotherapeutic treatment with cisplatin. PMID:19351554

Saad, Abir A; Youssef, Mokhtar I; El-Shennawy, Lamiaa K

2009-07-01

385

Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study.  

PubMed

Genomic DNA obtained from patient whole blood samples is a key element for genomic research. Advantages and disadvantages, in terms of time-efficiency, cost-effectiveness and laboratory requirements, of procedures available to isolate nucleic acids need to be considered before choosing any particular method. These characteristics have not been fully evaluated for some laboratory techniques, such as the salting out method for DNA extraction, which has been excluded from comparison in different studies published to date. We compared three different protocols (a traditional salting out method, a modified salting out method and a commercially available kit method) to determine the most cost-effective and time-efficient method to extract DNA. We extracted genomic DNA from whole blood samples obtained from breast cancer patient volunteers and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260/280 ratio and polymerase chain reaction product amplification) of the obtained yield. On average, all three methods showed no statistically significant differences between the final result, but when we accounted for time and cost derived for each method, they showed very significant differences. The modified salting out method resulted in a seven- and twofold reduction in cost compared to the commercial kit and traditional salting out method, respectively and reduced time from 3days to 1hour compared to the traditional salting out method. This highlights a modified salting out method as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples. PMID:22228086

Chacon-Cortes, Diego; Haupt, Larisa M; Lea, Rod A; Griffiths, Lyn R

2012-05-01

386

A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga  

PubMed Central

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP1011) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 g mg?1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae. PMID:24867404

Greco, Maria; Saez, Claudio A.; Brown, Murray T.; Bitonti, Maria Beatrice

2014-01-01

387

Efficient method for mycobacterial DNA extraction in blood cultures aids rapid PCR identification of Mycobacterium tuberculosis and Mycobacterium avium.  

PubMed

The study presented here evaluated the utility of several methods of extracting mycobacterial nucleic acids from positive blood culture samples and examined the effect of each method on the performance of an in-house PCR used directly in the peripheral blood of 80 patients with AIDS to identify Mycobacterium spp. The modified Boom method for extracting DNA from blood cultures proved to be the most efficient, with subsequent PCR analysis yielding 100% positivity (7 samples positive for M. avium and 5 for M. tuberculosis). Only three of 12 patients with a positive blood culture had a PCR result positive for M. avium in peripheral blood. The identification of mycobacteria by PCR in blood culture took about 3 days, reducing the time to diagnosis by several weeks. These results demonstrate that PCR is a sensitive and quick method for identifying mycobacteria, especially when a good DNA extraction method is applied. PMID:15558344

Nakatani, S M; Burger, M; Assef, M C; Brockelt, S R; Cogo, L L; Messias-Reason, I J T

2004-11-01

388

Technical note: A simple salting-out method for DNA extraction from milk somatic cells: investigation into the goat CSN1S1 gene.  

PubMed

In this study, a sensitive, rapid, and toxic solvent-free method to extract DNA from milk somatic cells was implemented for characterization of the goat alphaS1-casein gene (CSN1S1). Methods reported for purification of DNA from milk often involve organic extraction, overnight incubation, or use of expensive commercial kits. The present method was implemented for goat milk and is based on a salting-out protocol. The method yielded an amount of DNA suitable for PCR-RFLP without the need for sample enrichment. The PCR-RFLP of DNA extracted from milk produced amplified and digested products of correct size, comparable with those obtained using PCR-RFLP of DNA extracted from blood. Therefore, milk can be used as an alternative source of DNA, making sample collection easier and reducing stressful practices such as capture, handling, and venipuncture in animal management. PMID:17582139

d'Angelo, F; Santillo, A; Sevi, A; Albenzio, M

2007-07-01

389

Extraction of Plasmid DNA The majority of the work done with plasmids in M. tuberculosis strains is with plasmids  

E-print Network

to retrieve a plasmid from an M. tuberculosis transformant to verify the integrity of an area of interest of bacteria for plasmid extraction is based on the E. coli method pioneered by Birboim et al (Birnboim/mL 4. Chloroform:methanol (1:1) B. Protocol Steps 1. Prior to harvest (3 to 24 hours) add glycine

390

Rapid detection of Rhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method  

Microsoft Academic Search

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR)\\u000a was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract\\u000a parasite DNA from diseased

Masaru Matsumoto; Naruto Furuya; Yoichi Takanami; Nobuaki Matsuyama

1997-01-01

391

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development  

Microsoft Academic Search

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA\\u000a extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic\\u000a analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly\\u000a 62% of all DNA sequences analyzed.

Randy P. Revetta; Robin S. Matlib

2011-01-01

392