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1

One-Step Pathogen Specific DNA Extraction from Whole Blood on a Centrifugal Microfluidic Device  

Microsoft Academic Search

We could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood utilizing centrifugal microfluidics on a polymer based CD platform. By combining the TS-LIMBS (target separation and laser-irradiated magnetic bead system) and centrifugal microfluidics using the novel LIFM (the laser irradiated ferrowax microvalves), DNA extraction experiments from whole blood spiked with Hepatitis B virus

Yoon-Kyoung Cho; Jeong-Gun Lee; Jong-Myeon Park; Beom-Seok Lee; Youngsun Lee; Christopher Ko

2007-01-01

2

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).

2012-06-26

3

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.

Lana Hays

2009-01-01

4

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.

Lana Hays

2009-01-01

5

Yeast DNA Extraction  

NSDL National Science Digital Library

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

Lana Hays

2009-01-01

6

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.

Lana Hays

2009-01-01

7

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.

2007-02-02

8

Stepping stones in DNA sequencing  

PubMed Central

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics. PMID:22887891

Stranneheim, Henrik; Lundeberg, Joakim

2012-01-01

9

Exploring DNA Extraction Erica Butts  

E-print Network

(By adding a protease) DNA Binding (DNA Binds to magnetic or silica beads and is washed) Precipitation;Applied Genetics Definition of Relative Extraction Efficiency · Recovery compared to another method to precipitate DNA · Rehydrated with 100 µL TE #12;Applied Genetics Blood Extracted DNA Cells Extraction

Perkins, Richard A.

10

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.

Mission Science Workshop

2012-01-01

11

Lima Bean Bacteria DNA Extraction  

NSDL National Science Digital Library

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

Lana Hays

2009-01-01

12

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).

13

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.

University of Utah

2008-01-01

14

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

15

Automated DNA extraction from pollen in honey.  

PubMed

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

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

2014-04-15

16

DNA Extraction Techniques for Use in Education  

ERIC Educational Resources Information Center

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

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

2010-01-01

17

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

18

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

19

MATERIALS AND METHODS 1) DNA extraction  

E-print Network

MATERIALS AND METHODS 1) DNA extraction · DNA was extracted from the ileo-cecal nodes of 475 Holstein cows from two herds using the Qiagen DNA extraction kit (Valencia, CA). 2) Map detection · Map is estimated to be present in 67% of US dairy herds and results in significant economic loss. A 70kb region

Collins, Gary S.

20

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

E-print Network

these possibilities have yet to be explored by us. Notes on Contamination Control Since DNA extracted from ancientKemp Lab Ancient DNA Extraction Protocol- "New" Method Prepared by Brian M. Kemp, September 2012 of extreme loss of short DNA fragments associated with steps commonly employed in the extraction of ancient

Kemp, Brian M.

21

Bacterial and fungal DNA extraction from blood samples: automated protocols.  

PubMed

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA. PMID:25319785

Lorenz, Michael G; Disqu, Claudia; Mhl, Helge

2015-01-01

22

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.

23

Evaluation of DNA extraction methods from complex phototrophic biofilms.  

PubMed

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 +/- 12 to 1893 +/- 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol-chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol-chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms. PMID:20140796

Ferrera, Isabel; Massana, Ramon; Balagu, Vanessa; Pedrs-Ali, Carles; Snchez, Olga; Mas, Jordi

2010-04-01

24

Functions of base selection step in human DNA polymerase ?  

Microsoft Academic Search

Recent studies have revealed that the base selection step of DNA polymerases (pol) plays a role in prevention of DNA replication errors. We investigated whether base selection is required for the DNA replication fidelity of pol ? and genomic stability in human cells. We introduced an Leu864 to Phe substitution (L864F) into human pol ? and performed an in vitroLacZ?

Shigeru Tanaka; Ke Cao; Atsuko Niimi; Siripan Limsirichaikul; Huang Qin Miao; Noriko Nakamura; Takashi Murate; Yoshinori Hasegawa; Takashi Takahashi; Motoshi Suzuki

2010-01-01

25

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

26

An efficient method for genomic DNA extraction from different molluscs species.  

PubMed

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; Leito, Alexandra; Guedes-Pinto, Henrique

2011-01-01

27

Protocol: A high-throughput DNA extraction system suitable for conifers  

Microsoft Academic Search

BACKGROUND: High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA

Stanislav Bashalkhanov; Om P Rajora

2008-01-01

28

Force steps during viral DNA packaging ?  

E-print Network

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

Prashant K. Purohit; Jane' Kondev; Rob Phillips

2003-09-22

29

[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

30

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

31

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

32

DNA digestion to deoxyribonucleoside: A simplified one-step procedure  

PubMed Central

We present a simple and inexpensive one-step protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, thes new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol. PMID:18028864

Quinlivan, Eoin P.; Gregory, Jesse F.

2008-01-01

33

Comparison of DNA Extraction Methods from Small Samples of Newborn Screening Cards Suitable for Retrospective Perinatal Viral Research  

PubMed Central

Reliable detection of viral DNA in stored newborn screening cards (NSC) would give important insight into possible silent infection during pregnancy and around birth. We sought a DNA extraction method with sufficient sensitivity to detect low copy numbers of viral DNA from small punch samples of NSC. Blank NSC were spotted with seronegative EDTA-blood and seropositive EBV EDTA-blood. DNA was extracted with commercial and noncommercial DNA extraction methods and quantified on a spectrofluorometer using a PicoGreen dsDNA quantification kit. Serial dilutions of purified viral DNA controls determined the sensitivity of the amplification protocol, and seropositive EBV EDTA-blood amplified by nested PCR (nPCR) validated the DNA extraction methods. There were considerable differences between the commercial and noncommercial DNA extraction methods (P=0.014; P=0.016). Commercial kits compared favorably, but the QIamp DNA micro kit with an added forensic filter step was marginally more sensitive. The mean DNA yield from this method was 3 ng/?l. The limit of detection was 10 viral genome copies in a 50-?l reaction. EBV nPCR detection in neat and 1:10 diluted DNA extracts could be replicated reliably. We conclude that the QIamp Micro DNA extraction method with the added forensic spin-filter step was suitable for retrospective DNA viral assays from NSC. PMID:21455476

McMichael, Gai L.; Highet, Amanda R.; Gibson, Catherine S.; Goldwater, Paul N.; O'Callaghan, Michael E.; Alvino, Emily R.; MacLennan, Alastair H.

2011-01-01

34

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

2002-01-01

35

DNA  

NSDL National Science Digital Library

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

Doherty, Jennifer; Waldron, Ingrid

36

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

37

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

38

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

PubMed

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

Banaszak, Anastazia T

2007-01-01

39

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

PubMed Central

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

Zhou, Zhongwu; Kadam, Ulhas; Irudayaraj, Joseph

2014-01-01

40

Extraction of DNA from plant and fungus tissues in situ  

PubMed Central

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

2012-01-01

41

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

42

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

PubMed

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

Takakura, Koh-Ichi; Nishio, Takayuki

2012-11-01

43

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

44

Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts  

Technology Transfer Automated Retrieval System (TEKTRAN)

Accurate DNA concentration estimates from environmental samples using minimal sample volumes are essential for most downstream applications. To compare the efficacy of microvolume quantification methods, DNA was extracted from soil, compost, and pure culture samples, and quantified using two absorba...

45

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

46

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

47

One step DNA assembly for combinatorial metabolic engineering.  

PubMed

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity. PMID:24594279

Coussement, Pieter; Maertens, Jo; Beauprez, Joeri; Van Bellegem, Wouter; De Mey, Marjan

2014-05-01

48

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

PubMed

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

Lazarevic, Vladimir; Gaa, Nadia; Girard, Myriam; Franois, Patrice; Schrenzel, Jacques

2013-01-01

49

A novel method of genomic DNA extraction for Cactaceae1  

PubMed Central

Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

2013-01-01

50

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

PubMed Central

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

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

2014-01-01

51

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments  

PubMed Central

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (?-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting. PMID:19904232

Lee, Sangwon; Hallam, Steven J.

2009-01-01

52

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

E-print Network

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

Wurtele, Eve Syrkin

53

Automatable full demineralization DNA extraction procedure from degraded skeletal remains  

Microsoft Academic Search

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop

Sylvain Amory; Ren Huel; Ana Bili?; Odile Loreille; Thomas J. Parsons

54

High efficiency DNA extraction from bone by total demineralization  

Microsoft Academic Search

In historical cases, missing persons identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the

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

2007-01-01

55

EXTRACTION AND PURIFICATION OF MICROBIAL DNA FROM SEDIMENTS  

EPA Science Inventory

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

56

Cost-effective method of DNA extraction from taeniid eggs  

Microsoft Academic Search

A new cost-effective method using silicon dioxide- and guanidine isothiocyanate-containing buffers, after previous alkaline\\u000a lysis, was established for the DNA extraction from taeniid eggs isolated from canine faeces. The purified DNA can be used\\u000a to amplify the species-specific 12S mitochondrial DNA of Echinococcus multilocularis in direct and nested polymerase chain reaction in order to differentiate between E. multilocularis and Taenia

V. Dyachenko; E. Beck; N. Pantchev; C. Bauer

2008-01-01

57

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

58

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

59

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

PubMed

The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed. PMID:21864928

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

2011-11-01

60

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

61

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

Microsoft Academic Search

Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint

Leslie Cler; Dawei Bu; Cheryl Lewis; David Euhus

2006-01-01

62

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

PubMed

Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO()150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch()Forensic DNA Purification Kit (Invitrogen), the PrepFilerAutomated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. PMID:22310206

Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gbel, Gabriella; Rscheisen, Christiane

2012-09-01

63

DNA extraction protocol for rapid PCR detection of pathogenic bacteria.  

PubMed

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

Brewster, Jeffrey D; Paoli, George C

2013-11-01

64

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

65

Feature Extraction for DNA Microarray Data  

Microsoft Academic Search

In this study we perform wavelet analysis on high dimensional microarray data. We perform two methods of feature extraction on microarray data, using approximation coefficients and detail coefficients. A set of orthogonal wavelet approximation coefficients based on wavelet decomposition are extracted to compress the gene profiles and reduce the dimensionality of microarray data. A set of orthogonal wavelet detail coefficients

Yihui Liu

2007-01-01

66

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

PubMed

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

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

2014-04-01

67

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

PubMed Central

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

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

2014-01-01

68

Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments.  

PubMed

A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments. The odds of obtaining the correct plasmid clone in a single step, using conventional techniques, is less than 1 in 191000000. Using CRC, the desired plasmid was recovered at a frequency of one in two. Ligation is no longer the rate limiting step in cloning, and limitless possibilities exist for the reconstruction of complex genomes. PMID:10675609

Pachuk, C J; Samuel, M; Zurawski, J A; Snyder, L; Phillips, P; Satishchandran, C

2000-02-01

69

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

70

Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.  

PubMed

During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors. PMID:24331004

Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

2013-01-01

71

Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases  

PubMed Central

During the last 20years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors. PMID:24331004

Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

2013-01-01

72

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

E-print Network

reserved. doi:10.1016/j.jas.2006.02.015 Journal of Archaeological Science 33 (2006) 1680e1689 http to polymerase [11]. Inhibitors are also routinely en- countered in forensic investigations of DNA extracted from

Kemp, Brian M.

73

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: an alternative to microdialysis filtration.  

PubMed

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon() and Microcon() centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin() XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin() XS column is approximately 30 min vs. approximately 1.5h with the Centricon() YM-100 filter device. PMID:20457101

Hudlow, William R; Krieger, Robert; Meusel, Markus; Sehhat, Joshua C; Timken, Mark D; Buoncristiani, Martin R

2011-06-01

74

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

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

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

2012-05-01

75

One-step assay for the quantification of T4 DNA ligase.  

PubMed

As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5%) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays. PMID:25503935

Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

2015-02-01

76

Microfluidic DNA extraction using a patterned aluminum oxide membrane  

NASA Astrophysics Data System (ADS)

A DNA extraction system was designed and fabricated using an AOM (aluminum oxide membrane) with 200 nm pores and PDMS microfluidic channels. The membrane was patterned using soft lithography techniques and SU-8 photolithography on the membrane. After making the pattern with SU-8, the AOM was observed using an SEM (scanning electro microscope) to verify the AOM structure was not damaged. From the SEM images, the AOM structure was not different after modification with SU-8. To complete the system, a PDMS mold for the microfluidic channels was made by soft lithography. Using the SU-8 mold, PDMS microchannels were cast using PDMS with a low polymer to curing agent ratio to provide adhesion between the patterned membrane and microfluidic channel. Then, the patterned membrane was sandwiched between PDMS microfluidic channels in a parallel format. The completed system was tested with 10ug of Lambda DNA mixed with the fluorescent dye SYBR Green I. Following DNA extraction, the surface of each well was examined with fluorescence microscopy while embedded in the microfluidic system. Extracted and immobilized DNA on the AOM was observed in almost every separation well. This microsystem, referred to as a membrane-on-a-chip, has potential applications in high-throughput DNA extraction and analysis, with the possibility of being integrated into polymer-based microfluidic systems.

Kim, Jungkyu; Gale, Bruce K.

2006-01-01

77

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

78

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 a specific purpose in the process of extracting DNA. Scientists use a very similar process to isolate DNA

Rose, Michael R.

79

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

80

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

Microsoft Academic Search

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

Mohamad Ghazali; Jalan Sultan

81

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

82

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA*  

PubMed Central

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ?75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP. PMID:23596008

Ramanagoudr-Bhojappa, Ramanagouda; Chib, Shubeena; Byrd, Alicia K.; Aarattuthodiyil, Suja; Pandey, Manjula; Patel, Smita S.; Raney, Kevin D.

2013-01-01

83

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

84

Selective extraction of gold and platinum in water using ionic liquids. A simple two-step extraction process.  

PubMed

We report an all-ionic liquid process for the separation of tetrachloroaurate and hexachloroplatinate complexes. In a first step, gold is removed from water by liquid-liquid extraction with a hydrophobic ionic liquid, 1,2-dimethyl-3-octylimidazolium bis(trifluoromethylsulfonyl)imide. Platinum is subsequently extracted from the solution in the presence of KSCN using 1-methyl-3-octylimidazolium bis(trifluoromethylsulfonyl)imide. PMID:23250110

Papaiconomou, Nicolas; Gnand-Pinaz, Sbastien; Leveque, Jean-Marc; Guittonneau, Sylvie

2013-02-14

85

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.

86

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

PubMed Central

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

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

2014-01-01

87

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.  

PubMed

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 ?L of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 ?L of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 ?m mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman 903 paper, dried blood stains on FTA cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems. PMID:25070548

Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

2014-10-01

88

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

89

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods  

Microsoft Academic Search

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting phys- icochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal inter- genic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition

F. Martin-Laurent; L. Philippot; S. Hallet; R. Chaussod; J. C. Germon; G. Soulas; G. Catroux

2001-01-01

90

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

PubMed Central

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

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

2007-01-01

91

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

Microsoft Academic Search

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

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

2010-01-01

92

Forensic Strategies Used for DNA Extraction of Ancient and Degraded Museum Sturgeon Specimens  

Microsoft Academic Search

The procedure for the successful extraction of nucleic acids depends on the type of sample being extracted and the purpose\\u000a of the extraction. Protocols for the extraction of high copy number DNA samples vary significantly from those of degraded\\u000a DNA samples. This study involved the development of a DNA extraction protocol that includes a cleaning procedure designed\\u000a to remove external

E. Martinez-Espin; L. J. Martinez-Gonzalez; J. C. Alvarez; R. K. Roby; J. A. Lorente

93

Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.  

PubMed

Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction. PMID:15872286

Aldous, Wade K; Pounder, June I; Cloud, Joann L; Woods, Gail L

2005-05-01

94

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

PubMed

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

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

2015-04-15

95

Evaluation of four automated protocols for extraction of DNA from FTA cards.  

PubMed

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards. PMID:23574642

Stangegaard, Michael; Brsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

2013-10-01

96

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

PubMed Central

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

97

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR.  

PubMed

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions. PMID:21311936

van Tongeren, S P; Degener, J E; Harmsen, H J M

2011-09-01

98

Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.  

PubMed

The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight. PMID:25365545

Tomlinson, Christopher G; Moye, Aaron L; Holien, Jessica K; Parker, Michael W; Cohen, Scott B; Bryan, Tracy M

2015-01-15

99

'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

100

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

101

Protocol Cycle Time Optimization on a Microfluidic Cartridge for DNA\\/RNA Extraction Application  

Microsoft Academic Search

A disposable microfluidic cartridge for DNA\\/RNA extraction has been developed. Different reagents are used to bind and unbind DNA on the chip used in the cartridge. The extraction of DNA\\/RNA is based on a particular protocol, which involves the dispensing of different reagents at various flow rates. Dispensing of reagents is controlled by a needle valve which is integrated in

Michelle Chew; Ling Xie; CS Premachandran; Seung Wook Yoon

2007-01-01

102

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

103

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

104

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

105

Application of BioRobot M48 to forensic DNA extraction  

Microsoft Academic Search

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized

Agnieszka Parys-Proszek; Wojciech Branicki; Paulina Wola?ska-Nowak; Tomasz Kupiec

2008-01-01

106

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

107

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

108

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ system.  

PubMed

The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ system combined with the Zymo Research Mini RNA Isolation Kit II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain. PMID:20457058

Bowden, Anna; Fleming, Rachel; Harbison, SallyAnn

2011-01-01

109

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

110

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

PubMed

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

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

2014-01-01

111

Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles.  

PubMed

DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap. PMID:1060140

Summers, J; O'Connell, A; Millman, I

1975-11-01

112

Genome of Hepatitis B Virus: Restriction Enzyme Cleavage and Structure of DNA Extracted from Dane Particles  

Microsoft Academic Search

DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R\\\\cdot HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this

Jesse Summers; Anna O'Connell; Irving Millman

1975-01-01

113

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

PubMed

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

Mohandesan, Elmira; Prost, Stefan; Hofreiter, Michael

2012-01-01

114

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

PubMed

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859

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

2012-04-01

115

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

PubMed Central

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859

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

2012-01-01

116

Organizing and Searching the World Wide Web of Facts -Step One: the One-Million Fact Extraction Challenge  

E-print Network

of arbitrary quality. The extraction starts from as few as 10 seed facts, requires no ad- ditional input this case, facts), and iteratively grows it by finding contextual patterns that extract the seeds fromOrganizing and Searching the World Wide Web of Facts - Step One: the One-Million Fact Extraction

Yang, Junfeng

117

DNA Extraction by Isotachophoresis in a Microfluidic Channel  

SciTech Connect

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

Stephenson, S J

2011-08-10

118

Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA.  

PubMed

Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process. PMID:16889396

Freitas, S S; Santos, J A L; Prazeres, D M F

2006-01-01

119

HELICASE DEPENDENT AMPLICATION transfer the DNA  

E-print Network

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

120

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

PubMed

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

121

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

PubMed

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

Phillips, Kirsty; McCallum, Nicola; Welch, Lindsey

2012-03-01

122

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

123

Early Steps in the DNA Base Excision/Single-Strand Interruption Repair Pathway in Mammalian Cells  

PubMed Central

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 45 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3? OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3? phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified that may affect activity and complex formation. The focus of this review are the early steps in mammalian BER for oxidized damage. PMID:18166975

Hegde, Muralidhar L.; Hazra, Tapas K.; Mitra, Sankar

2009-01-01

124

Tenrec phylogeny and the noninvasive extraction of nuclear DNA.  

PubMed

Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses and disturbance of natural populations. PMID:16522569

Asher, Robert J; Hofreiter, Michael

2006-04-01

125

DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth  

PubMed Central

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

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

2013-01-01

126

A high-throughput, high-quality plant genomic DNA extraction protocol.  

PubMed

The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 g high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/L). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications. PMID:24222228

Li, H; Li, J; Cong, X H; Duan, Y B; Li, L; Wei, P C; Lu, X Z; Yang, J B

2013-01-01

127

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

128

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis  

PubMed Central

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials. PMID:10908366

Pang, Ho-ming; Yeung, Edward S.

2000-01-01

129

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

130

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

PubMed Central

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

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

2010-01-01

131

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

Microsoft Academic Search

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

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

1996-01-01

132

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...

133

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...

134

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ system  

Microsoft Academic Search

The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential

Anna Bowden; Rachel Fleming; SallyAnn Harbison

2011-01-01

135

A REVIEW OF CURRENT KNOWLEDGE OF TECHNIQUES TO EXTRACT AND AMPLIFY DNA FROM 'DIFFICULT' WHALE SAMPLES  

Microsoft Academic Search

DNA analysis methods currently used for whale product identification are dependent on extraction and PCR amplification of cetacean nucleic acids, but certain product types and intensive processing may restrict the amount of DNA recovered or degrade the DNA and inhibit amplification. Newly developed methods developed for \\

Frank Cipriano; Luis Pastene

2009-01-01

136

DNA Extraction 5th Grade Physical Science Standard 1.1  

E-print Network

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

137

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

138

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

E-print Network

will extract this DNA using dish soap, salt, water, a coffee filter and alcohol. Adult moderator should have of water) 1 coffee filter (use 2-4 filters for extra strength - be careful not to push pulp through or out can for 2 minutes. Pass the bag to Student 2. Student 2: Add 2 teaspoons of extraction liquid

Gruber, Jonathan

139

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

140

[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

141

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenolchloroformisoamyl alcohol DNA extraction  

PubMed Central

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

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

2015-01-01

142

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

PubMed

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

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

2015-01-01

143

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

PubMed Central

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 ureaformaldehyde 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

144

Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.  

PubMed Central

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction. Images PMID:8076611

Adachi, Y; Laemmli, U K

1994-01-01

145

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

PubMed Central

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

2014-01-01

146

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

147

The Elimination of DNA from the Cry Toxin-DNA Complex Is a Necessary Step in the Mode of Action of the Cry8 Toxin  

PubMed Central

Several crystal (Cry) proteins are known to occur as DNA-protein complexes. However, the role of the DNA associated with the activated toxin in the mechanism of action of the Cry toxin has long been ignored. Here, we focused on the DNA-activated Cry toxin complex. Both forms of the Cry8Ca2 and Cry8Ea1 toxins, i.e., with or without bound DNA, were separately obtained. Size-exclusion chromatography analysis indicated that the Cry8Ca2 toxin-DNA complex has a tight or compact structure. The Cry8Ca2 toxin-DNA complex is more likely to move toward the air/water interface and is more hydrophobic than the toxin without DNA. Competitive binding assays indicated that the Cry8Ca2 and Cry8Ea1 toxins without DNA specifically bind to the midgut of Anomala corpulenta and Holotrichia parallela larvae, respectively. In contrast, the association of DNA with each toxin might result in the nonspecific recognition of the Cry toxin and its target receptor in the insect midgut. The association of the DNA fragment with the Cry8 toxin was shown to protect the Cry protein from digestion by proteases. Based on our results, we propose an additional step in the mechanism of action of the Cry8 toxin and elucidate the function of the associated DNA as well as the importance of the removal of this DNA for the insecticidal activity of the toxin. PMID:24324685

Ai, Bingjie; Li, Jie; Feng, Dongmei; Li, Feng; Guo, Shuyuan

2013-01-01

148

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

149

DNA extract characterization process for microbial detection methods development and validation  

PubMed Central

Background Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. Results DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/?L. Forty-seven extracts had concentrations greater than 17.5 ng/?L, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ ?L), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/?L to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/?L and 0.25 ng/?L respectively. The qPCR LOQ varied by cell type (5.72??10-3 ng/?L for E. coli, 2.66??10-3 ng/?L, for B. cereus, 3.78??10-3 ng/?L for B. thailandensis, and 7.67??10-4 ng/?L for S. cerevisiae). A number of samples were below the UV spectroscopy (n?=?27), flurometry (n?=?15), and qPCR (n?=?3) LOQ. Conclusion The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics. PMID:23206278

2012-01-01

150

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

151

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

PubMed Central

Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil, PowerFecal, NucleoSpin Soil kits and QIAamp DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin Soil kit (NS kit), and to a lesser extent the PowerFecal kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

Desneux, Jrmy; Pourcher, Anne-Marie

2014-01-01

152

A new DNA extraction method by controlled alkaline treatments from consolidated subsurface sediments.  

PubMed

Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere. PMID:22092362

Kouduka, Mariko; Suko, Takeshi; Morono, Yuki; Inagaki, Fumio; Ito, Kazumasa; Suzuki, Yohey

2012-01-01

153

Evaluation of three methods for effective extraction of DNA from human hair.  

PubMed

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method. PMID:15866502

Suenaga, Emi; Nakamura, Hiroshi

2005-06-01

154

Viscum album L. Extracts Protects HeLa Cells against Nuclear and Mitochondrial DNA Damage  

PubMed Central

Viscum album L. is a semiparasitic plant grown on trees and widely used for the treatment of many diseases in traditional and complementary therapy. It is well known that some activities of Viscum album extracts are varied depending on the host trees, such as antioxidant, apoptosis-inducing, anticancer activities of the plant. The aim of the present study is to examine the comparative effects of methanolic extracts of V. album grown on three different host trees (locust tree, lime tree, and hedge maple tree) on H2O2-induced DNA damage in HeLa cells. Oxidative damage in mitochondrial DNA and two nuclear regions was assessed by QPCR assay. The cells were pretreated with methanolic extracts (10??g/mL) for 48?h, followed by the treatment with 750??M H2O2 for 1 hour. DNA damage was significantly induced by H2O2 while it was inhibited by V. album extracts. All extracts completely protected against nuclear DNA damage. While the extract from lime tree or white locust tree entirely inhibited mitochondrial DNA damage, that from hedge maple tree inhibited by only 50%. These results suggest that methanolic extracts of V. album can prevent oxidative DNA damage, and the activity is dependent on the host tree. PMID:22988477

nay-Uar, Evren; Erol, zlem; Kandemir, Ba?ak; Merto?lu, Elif; Karagz, Ali; Arda, Nazl?

2012-01-01

155

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

156

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

Microsoft Academic Search

Abstract. 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 purification,

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

157

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

158

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration  

Microsoft Academic Search

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon and Microcon centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be

William R. Hudlow; Robert Krieger; Markus Meusel; Joshua C. Sehhat; Mark D. Timken; Martin R. Buoncristiani

2011-01-01

159

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

160

A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis.  

PubMed

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4-6C) 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

2013-12-01

161

EXTRACTION OF HIGH QUALITY GENOMIC DNA FROM LATEX-CONTAINING PLANTS  

Technology Transfer Automated Retrieval System (TEKTRAN)

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. H...

162

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

Microsoft Academic Search

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

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

1999-01-01

163

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

164

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

165

PCR-based typing of DNA extracted from cigarette butts  

Microsoft Academic Search

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

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

1991-01-01

166

DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.  

PubMed

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples. PMID:24680125

Yano, Shizue; Honda, Katsuya; Kaminiwa, Junko; Nishi, Takeki; Iwabuchi, Yayoi; Sugano, Yukiko; Kurosu, Akira; Suzuki, Yasuhito

2014-05-01

167

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

E-print Network

of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle researchPreparation and use of Xenopus egg extracts to study DNA replication and chromatin associated online 19 April 2012 Communicated by Marcel Mechali Keywords: Xenopus Egg extract In vitro Cell

Blow, J. Julian

168

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

PubMed

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

Smrekar, Vida; Smrekar, Franc; Strancar, Ale; Podgornik, Ale

2013-02-01

169

Two-steps extraction of essential oil, polysaccharides and biphenyl cyclooctene lignans from Schisandra chinensis Baill fruits.  

PubMed

A method for two-steps extraction of essential oil, polysaccharides and lignans from Schisandra chinensis Baill had been established. Firstly, S. chinensis was extracted by hydro-distillation, the extracted solution was separated from the water-insoluble residue and precipitated by adding dehydrated alcohol after the essential oil was collected, and then the precipitate as polysaccharide was collected. Finally, second extraction was performed to obtained lignans from the water-insoluble residue with ultrasonic-microwave assisted extraction (UMAE) method. Response surface methodology was employed to optimize the UMAE parameters, the optimal conditions were as follows: microwave power 430W, ethanol concentration 84%, particle size of sample 120-mesh sieves, ratio of water to raw material 15 and extraction time 2.1min. Under these optimized conditions, the total extraction yields of five lignans (Schisandrol A, Schisantherin A, Deoxyschisandrin, Schisandrin B and Schisandrin C) had reached 14.220.135mg/g. Compared with the traditional method of direct extraction of different bioactive components in respective procedure, the extraction yields of polysaccharides and the five lignans had reached 99% and 95%, respectively. The mean recoveries of the 5 lignan compounds and polysaccharides were 97.75-101.08% and their RSD value was less than 3.88%.The approach proposed in this study not only improved the extraction yield of lignans, but also elevated the utilization of Schisandra resources. PMID:24755113

Cheng, Zhenyu; Yang, Yingjie; Liu, Yan; Liu, Zhigang; Zhou, Hongli; Hu, Haobin

2014-08-01

170

New efficient DNA extraction method to access the microbiome of Ricinus communis seeds.  

PubMed

Ricinus communis (castor bean) seeds are used to produce an alcohol-soluble oil that is used in more than 400 industrial processes. Despite its economic importance, there has been little research on the endophytic microbiota of castor bean seeds. This microbiota is important for plant metabolic processes and may have considerable biotechnological potential, such as production of lipases and plant growth promoter agents. We evaluated several DNA extraction methodologies in order to access the microbial diversity of castor bean through a metagenomic approach. Based on our observations, we developed a new methodology that takes advantage of the low solubility of calcium phosphates and the high affinity of these phosphates for proteins and polysaccharides. The extracted DNA quality was evaluated by PCR, using a selective primer pair for bacterial and mitochondrial 16S rDNA genes (799F and 1492R). We found this methodology quantitatively and qualitatively more efficient than the other approaches. In evaluating this new extraction methodology, we found that the difficulties of DNA extraction from castor bean seeds, such as abundant oil, polysaccharides, phenolic compounds, and plant enzymes, could be overcome. The resulting extracts had high concentration and purity, and they were obtained faster than with previous methods. The samples contained virtually all of the DNA, including the microbial DNA; this was validated by PCR analysis. PMID:23479168

Santos, C D; Dias, A C C; Amaral, I M R; Bonetti, A M; Campos, T A

2013-01-01

171

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

172

A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification.  

PubMed

Accurate identification of fungal pathogens using a sequence-based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested. PMID:24865530

Romanelli, A M; Fu, J; Herrera, M L; Wickes, B L

2014-10-01

173

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

PubMed

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

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

2013-05-01

174

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

PubMed Central

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning. PMID:9358185

Aljanabi, S M; Martinez, I

1997-01-01

175

DNA in ancient bone - where is it located and how should we extract it?  

PubMed

Despite the widespread use of bones in ancient DNA (aDNA) studies, relatively little concrete information exists in regard to how the DNA in mineralised collagen degrades, or where it survives in the material's architecture. While, at the macrostructural level, physical exclusion of microbes and other external contaminants may be an important feature, and, at the ultrastructural level, the adsorption of DNA to hydroxyapatite and/or binding of DNA to Type I collagen may stabilise the DNA, the relative contribution of each, and what other factors may be relevant, are unclear. There is considerable variation in the quality of DNA retrieved from bones and teeth. This is in part due to various environmental factors such as temperature, proximity to free water or oxygen, pH, salt content, and exposure to radiation, all of which increase the rate of DNA decay. For example, bone specimens from sites at high latitudes usually yield better quality DNA than samples from temperate regions, which in turn yield better results than samples from tropical regions. However, this is not always the case, and rates of success of DNA recovery from apparently similar sites are often strikingly different. The question arises as to whether this may be due to post-collection preservation or just an artefact of the extraction methods used in these different studies? In an attempt to resolve these questions, we examine the efficacy of DNA extraction methods, and the quality and quantity of DNA recovered from both artificially degraded, and genuinely ancient, but well preserved, bones. In doing so we offer hypotheses relevant to the DNA degradation process itself, and to where and how the DNA is actually preserved in ancient bone. PMID:21855309

Campos, Paula F; Craig, Oliver E; Turner-Walker, Gordon; Peacock, Elizabeth; Willerslev, Eske; Gilbert, M Thomas P

2012-01-20

176

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

177

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods  

PubMed Central

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years. PMID:11319122

Martin-Laurent, F.; Philippot, L.; Hallet, S.; Chaussod, R.; Germon, J. C.; Soulas, G.; Catroux, G.

2001-01-01

178

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

PubMed Central

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

2010-01-01

179

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

PubMed

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

Hervella, Montserrat; Iiguez, Maitane G; Izagirre, Neskuts; Anta, Alberto; de-la-Ra, Concepcin

2015-01-01

180

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

181

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

PubMed

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

2014-02-01

182

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

183

An Efficient DNA Extraction Method for Desert Calligonum Species  

Microsoft Academic Search

Genetic conservation programs in arid environments rely on molecular methods for diversity assessments. DNA-based molecular\\u000a 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\\u000a exceptionally large amount of oxidized polyphenolic compounds, polysaccharides, and

Raoudha Abdellaoui; Hassen Gouja; Amel Sayah; Mohamed Neffati

184

Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses  

PubMed Central

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample. PMID:11872453

Weinbauer, Markus G.; Fritz, Ingo; Wenderoth, Dirk F.; Hfle, Manfred G.

2002-01-01

185

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...

186

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

NASA Astrophysics Data System (ADS)

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

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

2013-09-01

187

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

PubMed

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

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

2014-12-01

188

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

PubMed Central

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

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

2014-01-01

189

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

PubMed Central

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

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

2014-01-01

190

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

191

Exploring microbial diversity in volcanic environments: a review of methods in DNA extraction.  

PubMed

The last decade has been marked by a large number of studies focused on understanding the distribution of microorganisms in volcanic environments. These studies are motivated by the desire to elucidate how the geochemically extreme conditions of such environments can influence microbial diversity both on the surface and in the subsurface of the Earth. The exploration of microbial community diversity has generally not relied on culture-dependent methods, but has been carried out using environmental DNA extraction. Because of the large diversity of chemically and physically complex samples, extracting DNA from volcanic environments is technically challenging. In view of the emerging literature, and our own experience in the optimisation of methods for DNA extraction from volcanic materials, it is timely to provide a methodological comparison. This review highlights and discusses new insights and methods published on DNA extraction methods from volcanic samples, considering the different volcanic environments. A description of a recent method for DNA extraction from basalt and obsidian glass rock samples from Iceland is included. Finally, we discuss these approaches in the wider context of modern work to understand the microbial diversity of volcanic environments. PMID:17540467

Herrera, Aude; Cockell, Charles S

2007-07-01

192

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

193

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

E-print Network

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

Hoehl, Melanie Margarete

194

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

195

Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.  

PubMed

Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 ?g/?L and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk. PMID:25218756

Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

2014-11-01

196

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

197

The assessment of the geometry of dinucleotide steps in double-helical DNA; a new local calculation scheme.  

PubMed

In this paper, we develop a new local Euler-angle-based scheme for assessing the internal kinematics or geometry of a general dinucleotide step in double-helical DNA. The geometry of a dinucleotide step is completely defined by: (1) the base-pair parameters that describe the relative position and orientation of one base with respect to the other in a standard Watson-Crick base-pair, and (2) the step parameters that describe the relative position and orientation of the two base-pairs. The key feature of our scheme is that it makes use of the concept of a mid-step reference frame. In addition to ensuring that identical values of step parameters are obtained irrespective of the direction of reckoning of a dinucleotide step (in the 5'-->3' direction along either strand), this mid-step-triad concept leads to local definitions of the step parameters that render them independent of the overall global conformation of the oligomer in question. In addition to presenting our own calculation scheme we also examine critically the most widely used package for the calculation of some of the step and base-pair parameters, viz, the NEWHELIX suite of programmes by R.E. Dickerson. Finally, a dodecamer, a decamer and an octamer are arbitrarily selected from a public data-base (N.D.B at Rutgers), and their step parameters are calculated by using both NEWHELIX and the proposed scheme. A comparison of the results is given whereby it is shown that for the step parameters: Helical Twist and Slide, and the base-pair parameters Propeller and Buckle, NEWHELIX and our proposed scheme give rather similar values. Substantial differences are seen, however, in the case of Rise. Two alternative definitions are given by NEWHELIX for the calculation of Roll and Tilt. Whereas one definition agrees well with our proposed scheme, the other is substantially different. PMID:7666417

el Hassan, M A; Calladine, C R

1995-09-01

198

Tenrec Phylogeny and the Noninvasive Extraction of Nuclear DNA  

Microsoft Academic Search

Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application

Robert J. Asher; Michael Hofreiter

2006-01-01

199

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

200

Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.  

PubMed

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, ?-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out. PMID:25444238

Alvarez-Aldana, Adalucy; Martnez, Jos William; Seplveda-Arias, Juan C

2014-11-01

201

Immune responses to foot-and-mouth disease DNA vaccines can be enhanced by coinjection with the Isatis indigotica extract.  

PubMed

The Isatis indigotica extract is widely used in clinical practice for the treatment of influenza, epidemic hepatitis, epidemic encephalitis B etc. The goal of this study was to investigate whether coinjection of the Isatis indigotica extract with foot-and-mouth disease virus (FMDV) DNA vaccine could increase the protective immune response. Mice were vaccinated twice with either FMDV DNA vaccine plus the Isatis indigotica extract or DNA vaccine alone. Compared with the group of DNA vaccine alone, in the group that received DNA vaccine plus the Isatis indigotica extract was observed a significant increase in not only FMDV-specific antibody response but also T cell proliferation assay. All swine immunized with either FMDV DNA vaccine plus the Isatis indigotica extract or DNA vaccine alone were partially protected from FMDV challenge, but the signs of DNA vaccine plus the Isatis indigotica extract group were less severe than that of DNA vaccine alone. Coinjection of the Isatis indigotica extract with DNA vaccine has adjuvant effect on the immune response against viral pathogens and its application provides an effective strategy to improve the efficacy of DNA vaccines. PMID:15920343

Chen, Liang; Lin, Tao; Zhang, Hongxin; Su, Yongbo

2005-01-01

202

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

PubMed

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species. PMID:17469067

Ribeiro, R A; Lovato, M B

2007-01-01

203

A high throughput DNA extraction method with high yield and quality  

PubMed Central

Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need. PMID:22839646

2012-01-01

204

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

205

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

206

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

207

How to open the treasure chest? Optimising DNA extraction from herbarium specimens.  

PubMed

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

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

2012-01-01

208

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

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

2012-01-01

209

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

Microsoft Academic Search

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

I. Pfeiffer; I. Vlkel; H. Tubert; B. Brenig

2004-01-01

210

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

Microsoft Academic Search

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

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

2011-01-01

211

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

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

212

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

213

Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts  

PubMed Central

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

Knipscheer, Puck; Rschle, Markus; Schrer, Orlando D.; Walter, Johannes C.

2014-01-01

214

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

215

Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.  

PubMed

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

216

Integration of cell lysis, protein extraction, and digestion into one step for ultrafast sample preparation for phosphoproteome analysis.  

PubMed

Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient. It is expected to have broad applications in phosphoproteomics analysis. PMID:24958348

Liu, Fangjie; Ye, Mingliang; Pan, Yanbo; Zhang, Yi; Bian, Yangyang; Sun, Zhen; Zhu, Jun; Cheng, Kai; Zou, Hanfa

2014-07-15

217

One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA.  

PubMed

A straightforward electrochemical DNA biosensing approach based on exploiting organometallic compound, aminoferrocene (AFC), as electroactive probes was firstly demonstrated, where the probes could be directly labeled to the free phosphate groups of the hybridized PNA/DNA heteroduplexes merely through one-step conjugation in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. Briefly, mercapto-terminated peptide nucleic acid (PNA) was firstly immobilized onto gold electrode and used as the capture probes for the specific recognition of target single-stranded DNA (ssDNA). After hybridization, AFC probes were directly labeled to the free 5'-terminal phosphate groups, which were activated by EDC and imidazole, of the hybridized PNA/DNA heteroduplexes, and then they were exploited as the electroactive probes to monitor the hybridization. As the captured ssDNA was labeled with AFC in the stoichiometric ratio of 1:1, thus the electrochemical analysis of the proportionally labeled AFC based on differential pulse voltammetry (DPV) enabled a quantitative determination of sequence-specific DNA. Under optimal conditions, the approach presented a good linear relationship between the current intensities and logarithm of ssDNA concentrations in the range from 0.1nM to 100nM with a detection limit of 93pM, and it rendered satisfactory analytical performance in serum samples. Furthermore, it exhibited excellent specificity toward single-nucleotide polymorphism (SNP) and precluded complicated protocols. More importantly, the simplicity of this approach together with its compatibility with standard micro-fabrication techniques makes it great potential in practical applications, especially in microarray areas where simple procedures are preferred. PMID:25461140

Hu, Qiong; Deng, Xianbao; Yu, Xuehua; Kong, Jinming; Zhang, Xueji

2014-10-21

218

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

219

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

PubMed

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

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

2012-01-01

220

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

PubMed

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

Wang, Xiaohong; Sui, Shaochun

2011-06-01

221

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments  

Microsoft Academic Search

Aims: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. Methods and Results: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS,

S. Giacomazzi; F. Leroi; J.-J. Joffraud

2005-01-01

222

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

Microsoft Academic Search

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

Liang Chen; Tao Lin; HongXin Zhang; YongBo Su

2005-01-01

223

Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.  

PubMed

This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner. PMID:21034773

Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

2011-01-01

224

Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species.  

PubMed

When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O(2)(-), 89% (AE) and 94% (HE) (P<0.05), and (ii) (.)OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu(2+)/H(2)O(2), 94% (AE) and 100% (HE) (P<0.05), and (ii) SnCl(2), 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu(2+) could have also been triggered by other mechanisms. PMID:22005340

Fernandes, Andria da Silva; Mazzei, Jos Luiz; de Alencar, Alexandre Santos; Evangelista, Heitor; Felzenszwalb, Israel

2011-01-01

225

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

E-print Network

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

Steel, BradleyC.

226

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

227

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.  

PubMed

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes. PMID:21499752

Yoshikawa, Hisao; Dogruman-Al, Funda; Dogruman-Ai, Funda; Turk, Songul; Kustimur, Semra; Balaban, Neriman; Sultan, Nedim

2011-10-01

228

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

PubMed

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

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

2015-02-01

229

Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction.  

PubMed

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ?10? CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis. PMID:21546201

Dauphin, Leslie A; Walker, Roblena E; Petersen, Jeannine M; Bowen, Michael D

2011-07-01

230

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.  

PubMed

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC. PMID:20730710

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-01-01

231

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

232

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

PubMed

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

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

2010-06-01

233

GENESUS: a two-step sequence design program for DNA nanostructure self-assembly.  

PubMed

DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure. PMID:24724843

Tsutsumi, Takanobu; Asakawa, Takeshi; Kanegami, Akemi; Okada, Takao; Tahira, Tomoko; Hayashi, Kenshi

2014-01-01

234

Evaluation of Silica Resins for Direct and Efficient Extraction of DNA from Complex Biological Matrices in a Miniaturized Format  

Microsoft Academic Search

For DNA purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (?SPE)

Huijun Tian; Andreas F. R. Hhmer; James P. Landers

2000-01-01

235

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

PubMed

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

2012-05-01

236

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

237

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

E-print Network

­4). Based on an x-ray crystal- lographic structure of uracil-DNA glycosylase (UDG) (2), it was proposed that irreversibly to an active-site cysteine (Cys-145; Fig. 1a). This uncommon direct damage reversal mechanism

Dinner, Aaron

238

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

239

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

PubMed Central

We study the kinetics of the overstretching transition in ?-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.52 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85nm, indicating a cooperativity of ?25 basepairs. This mechanism increases the free energy for the elementary reaction to ?94 kBT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches. PMID:21843477

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

2011-01-01

240

DNA Fingerprinting in a Forensic Teaching Experiment  

ERIC Educational Resources Information Center

This article presents an experiment designed to provide students, in a classroom laboratory setting, a hands-on demonstration of the steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analysis of the data. This experiment demonstrates how DNA fingerprinting is performed and how long it takes. It

Wagoner, Stacy A.; Carlson, Kimberly A.

2008-01-01

241

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples  

PubMed Central

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals. PMID:25635817

Psifidi, Androniki; Dovas, Chrysostomos I.; Bramis, Georgios; Lazou, Thomai; Russel, Claire L.; Arsenos, Georgios; Banos, Georgios

2015-01-01

242

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

243

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

244

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

245

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

PubMed

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

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

2014-04-01

246

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

PubMed

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

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

2015-02-10

247

Rapid DNA Extraction from Dried Blood Spots on Filter Paper: Potential Applications in Biobanking  

PubMed Central

Objectives Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. Methods The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40?L blood was used. Results DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. Conclusion Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage. PMID:25562044

Choi, Eun-Hye; Lee, Sang Kwang; Ihm, Chunhwa; Sohn, Young-Hak

2014-01-01

248

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

249

Two-dimensional continuous wavelet transform algorithm for phase extraction of two-step arbitrarily phase-shifted interferograms  

NASA Astrophysics Data System (ADS)

By virtue of the anti-noise and anti-defect abilities, in this paper, a two-dimensional continuous wavelet transform algorithm is proposed to analyze two-step arbitrarily phase-shifted interferograms with an orthonormalization process. The novel algorithm takes the advantages of the two existing ones, and it has a remarkable ability to accurately and automatically extract full-field phase distribution from two phase-shifted interferograms where they may contain arbitrary and unknown phase shift, complex fringes with phase ambiguity, large fringe-frequency variations, noise, defects and corrupted fringes. The validity of the algorithm is demonstrated by both computer simulation and real experiments.

Ma, Jun; Wang, Zhaoyang; Pan, Tongyan

2014-04-01

250

The Second Step of ATP Binding to DnaK Induces Peptide Release  

Microsoft Academic Search

The interaction of the nucleotide-free molecular chaperone DnaK (Hsp70) fromEscherichia coliwith nucleotides was studied under equilibrium and transient kinetic conditions. These studies used the intrinsic fluorescence signal of the single tryptophan residue (Trp102) of DnaK, or of novel fluorescent nucleotide analogs of ADP and ATP, N8-(4-N?-methylan thraniloylaminobutyl)-8-aminoadenosine 5?-di- or triphosphate (MABA-ADP and MABA-ATP) as spectroscopic probes. Titration of MABA-ADP with

Holger Theyssen; Hans-Peter Schuster; Lars Packschies; Bernd Bukau; Jochen Reinstein

1996-01-01

251

Extraction of high-quality host DNA from feces and regurgitated seeds: a useful tool for vertebrate ecological studies.  

PubMed

DNA extraction methods for genotyping non-invasive samples have led to great advances in molecular research for ecological studies, and have been particularly useful for analyzing threatened species. However, scarce amounts of fragmented DNA and the presence of Taq polymerase inhibitors in non-invasive samples are potential problems for subsequent PCR amplifications. In this study we describe a novel technique for extracting DNA from alimentary tract cells found on external surfaces of feces and regurgitated seeds. The presence of contaminants and inhibitors is minimized and samples are preserved intact for use in other ecological research (e.g. trophic studies). The amplification efficiency and purity of the extracted DNA from feces were significantly higher than in commonly used extraction procedures. Moreover, DNA of two bird species was identified from seeds expelled by regurgitation. Therefore, this method may be suitable for future ecological studies of birds, and other vertebrate groups. PMID:19746259

Marrero, Patricia; Fregel, Rosa; Cabrera, Vicente M; Nogales, Manuel

2009-01-01

252

Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues  

PubMed Central

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE. PMID:24688314

Sengven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

253

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

254

DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees.  

PubMed

Understanding the root distribution of trees by soil coring is time -: consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m(-2)) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23-28 C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R(2) = 0.9307, P < 0.001) with the dry matter (g m(-2)) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

Bithell, Sean L; Tran-Nguyen, Lucy T T; Hearnden, Mark N; Hartley, Diana M

2014-01-01

255

In Vitro Effect of Aqueous Extract and Fraction IV Portion of Ximenia americana Stem Bark on Trypanosoma congolense DNA  

PubMed Central

Trypanosomosis is a debilitating disease affecting mainly livestock and humans in tropical Africa. Chemically synthesized drugs and medicinal plants have been used in the treatment and control of this disease. In this study, the in vitro effect of aqueous extracts and fraction IV extract of Ximenia americana stem bark on Trypanosoma congolense DNA was investigated. The extracts were incubated with the parasites in vitro at 300?mg/mL aqueous extract and 25?mg/mL fraction IV portion for 30, 60, and 120 mins. The DNA of the trypanosomes was isolated and digested using ECOR1 enzyme and subsequently PCR was carried out. Results showed that aqueous extract and fraction IV portion immobilized 55% and 90% of the trypanosomes after 30-minute incubation. Subsequent isolation of the parasite DNA and agarose gel electrophoresis did not reveal that cell death was as a result of DNA fragmentation. This suggests that cell death was by another mechanism of action. PMID:24587898

Maikai, Victor Ambrose; Maikai, Beatty Viv; Kobo, Patricia Ishyaku

2014-01-01

256

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

E-print Network

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 21, 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 m 2 g 21) 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

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

2011-01-01

257

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

258

A new improved method for extraction of DNA from teeth for the analysis of hypervariable loci.  

PubMed

A new method for better recovery of DNA suitable for amplification of hypervariable loci from fragments of teeth, consisting of two steps-scraping and aspiration, and extensive decalcification-is reported. Higher yields of high molecular weight DNA were obtained from the root, pulp, and crown of all kinds of 120 teeth, irrespective of gender, age, and source of teeth. HLA DQA1, 5 poly markers (LDLR, GYPA, HBGG, D7S8, and Gc), and other 12 short tandem repeat loci (HPRTB, F13B, LPL, D13S317, D7S820, D5S818, D21S11, D18S51, FGA, D8S1179, D3S1358, and vWA) could be successfully amplified and typed from recovered DNA. PMID:12040267

Trivedi, R; Chattopadhyay, P; Kashyap, V K

2002-06-01

259

Extracts from cigarette smoke induce DNA damage and cell adhesion molecule expression through different pathways.  

PubMed

Cigarette smoke is a major risk factor for human diseases, such as lung cancer and atherosclerosis. The present study was undertaken to investigate the effect of non-fractionated water-soluble cigarette smoke extract (NFWS CSE) on DNA damage and cellular adhesion molecule expression in human umbilical vein endothelial cells (HUVECs). DNA damage and the surface expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin were determined by the use of the comet assay and flow cytometry, respectively. NFWS CSE-induced DNA damage in a dose-dependent manner during a 2 h exposure. Pretreatment with ascorbic acid or alpha-tocopherol completely inhibited the NFWS CSE-induced DNA damage. NFWS CSE exposure also up-regulated the surface expression of ICAM-1 and E-selectin in HUVECs. Pretreatment with ascorbic acid or alpha-tocopherol had no effect on NFWS CSE-induced E-selectin and ICAM-1 expression. In contrast, the non-antioxidant metal chelator 1,10-phenanthroline partially suppressed the surface expression of ICAM-1 and E-selectin. These results suggest that NFWS CSE exposure induces both DNA damage and the surface expression of adhesion molecules in HUVECs. However, the molecular mechanism of these effects may be through different pathways: reactive oxygen species are involved in NFWS CSE-induced DNA damage but have little relation to NFWS CSE-induced E-selectin and ICAM-1 expression. PMID:15560890

Chen, Haw-Wen; Chien, Miao-Lin; Chaung, Yu-Han; Lii, Chong-Kuei; Wang, Tsu-Shing

2004-12-01

260

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

261

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

El Bali, Latifa; Diman, Aurlie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

2014-01-01

262

An integrated disposable device for DNA extraction and helicase dependent amplification  

Microsoft Academic Search

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on\\u000a samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world\\u000a health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated.\\u000a Bacteria lysis, nucleic acid extraction, and DNA amplification with a

Madhumita Mahalanabis; Hussam ALMuayad; Jane Y. Zhang; Catherine M. Klapperich

2010-01-01

263

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples  

Microsoft Academic Search

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic\\u000a methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits\\u000a have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial\\u000a kits.

Hisao Yoshikawa; Funda Dogruman-AI; Songul Turk; Semra Kustimur; Neriman Balaban; Nedim Sultan

264

Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns  

Microsoft Academic Search

Polyvinylpolypyrrolidone spin columns were used to rapidly purify crude soil DNA extracts from humic materials for polymerase chain reaction (PCR) analysis. The PCR detection limit for the tfdC gene, encoding chlorocatechol dioxygenase from the 2,4-dichlorophenoxyacetic acid degradation pathway, was 101?102 cells\\/g soil in inoculated soils. The procedure could be applied to the amplification of biodegradative genes from indigenous microbial populations

Marc Berthelet; Lyle G. Whyte; Charles W. Greer

1996-01-01

265

DNA Extraction-Free Quantification of Dehalococcoides spp. in Groundwater Using a Hand-Held Device.  

PubMed

Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(-1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates. PMID:25360694

Stedtfeld, Robert D; Stedtfeld, Tiffany M; Kronlein, Maggie; Seyrig, Gregoire; Steffan, Robert J; Cupples, Alison M; Hashsham, Syed A

2014-11-13

266

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

PubMed Central

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies. PMID:22514653

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

2012-01-01

267

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

PubMed Central

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

268

Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.  

PubMed

We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern. PMID:21256887

Twe, Stefanie; Wallisch, Stefanie; Bannert, Andrea; Fischer, Doreen; Hai, Brigitte; Haesler, Felix; Kleineidam, Kristina; Schloter, Michael

2011-03-01

269

A simple and efficient method for extraction of PCR-amplifiable DNA from chicken eggshells.  

PubMed

Recently, we reported a method for discriminating a Japanese brand of chicken, the Hinai-jidori. As an application of this method for discriminating Hinai-jidori eggs, we here report an efficient method for extracting maternal DNA from eggshells. Eggshell powder was completely decalcified with EDTA solution, and then DNA was isolated by conventional phenol-chloroform extraction and ethanol precipitation. The efficiency of DNA recovery from eggshells was 50-fold higher than that of a previously reported method. The recovered DNA could be used for PCR, and 10 markers for identifying the Hinai-jidori chicken were detected. The genotypes of the Hinai-jidori exactly matched those of the Hinai-dori breed. Using this method, Hinai-jidori and Hinai-dori eggs could be distinguished from the eggs of Rhode Island Reds. This is the first report of a technique that can be used to discriminate the eggs of Hinai-jidori from those of other chickens, and it can also be utilized to validate the labeling of Hinai-jidori eggs in the market. PMID:20163594

Rikimaru, Kazuhiro; Takahashi, Hideaki

2009-04-01

270

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

PubMed Central

This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitts lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC50<0.01 g/ml) with a high therapeutic index (>28 000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC50=2.9 g/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1a was most active in reducing the cell-free EBV DNA (EC50=1.38 g/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV. PMID:21528487

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

2011-01-01

271

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

272

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

273

Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma  

PubMed Central

Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35?GE/mL versus 259,43?GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe. PMID:24222898

Ordoez, Elena; Rueda, Laura; Caadas, M. Paz; Fuster, Carme; Cirigliano, Vincenzo

2013-01-01

274

An Easy, Rapid, and Cost-Effective Method for DNA Extraction from Various Lichen Taxa and Specimens Suitable for Analysis of Fungal and Algal Strains  

PubMed Central

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. PMID:25606001

Park, Sook-Young; Jang, Seol-Hwa; Oh, Soon-Ok; Kim, Jung A

2014-01-01

275

Validation of the Isotropic Fractionator: Comparison with Unbiased Stereology and DNA Extraction for Quantification of Glial Cells  

PubMed Central

Background The isotropic fractionator (IF) is a novel cell counting technique that homogenizes fixed tissue, recovers cell nuclei in solution, and samples and quantifies nuclei by extrapolation. Studies using this technique indicate that the ratio of glia to neurons in the human brain is approximately 1:1 rather than the 10:1 or 50:1 ratio previously assumed. Although some results obtained with the IF have been similar to those obtained by stereology, the IF has never been calibrated or validated. It is conceivable that only a fraction of glial cell nuclei are recovered intact or recognized after the homogenization step. New Method To rule out this simple explanation for the claim of a 1:1 glia-neuron ratio, we compared cell numbers obtained from adjacent, weight-normalized samples of human and macaque monkey white matter using three techniques: the IF, unbiased stereology of histological sections in exhaustively sectioned samples, and cell numbers calculated from DNA extraction. Results and comparison of methods In primate forebrains, the IF yielded 73,00090,000 nuclei/mg white matter, unbiased stereology yielded 75,00092,000 nuclei/mg, with coefficients of error ranging from 0.0130.063, while DNA extraction yielded only 4,00023,000 nuclei/mg in fixed white matter tissues. Conclusions Since the IF revealed about 100% of the numbers produced by unbiased stereology, there is no significant underestimate of glial cells. This confirms the notion that the human brain overall contains glial cells and neurons with a ratio of about 1:1 far from the originally assumed ratio of 10:1 in favor of glial cells. PMID:24239779

Bahney, Jami; von Bartheld, Christopher S.

2014-01-01

276

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

PubMed

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

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

2012-03-01

277

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response.  

PubMed

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage. PMID:25169698

Dantuma, Nico P; Acs, Klara; Luijsterburg, Martijn S

2014-11-15

278

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

279

Sources of Pre-Analytical Variations in Yield of DNA Extracted from Blood Samples: Analysis of 50,000 DNA Samples in EPIC  

PubMed Central

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies. PMID:22808065

Caboux, Elodie; Lallemand, Christophe; Ferro, Gilles; Hmon, Bertrand; Mendy, Maimuna; Biessy, Carine; Sims, Matt; Wareham, Nick; Britten, Abigail; Boland, Anne; Hutchinson, Amy; Siddiq, Afshan; Vineis, Paolo; Riboli, Elio; Romieu, Isabelle; Rinaldi, Sabina; Gunter, Marc J.; Peeters, Petra H. M.; van der Schouw, Yvonne T.; Travis, Ruth; Bueno-de-Mesquita, H. Bas; Canzian, Federico; Snchez, Maria-Jos; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Nria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Franoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre

2012-01-01

280

Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening  

PubMed Central

Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ?1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ?98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS. PMID:24498621

Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

2013-01-01

281

Protection by Salvia extracts against oxidative and alkylation damage to DNA in human HCT15 and CO115 cells.  

PubMed

DNA damage induced by oxidative and alkylating agents contributes to carcinogenesis, leading to possible mutations if replication proceeds without proper repair. However, some alkylating agents are used in cancer therapy due to their ability to induce DNA damage and subsequently apoptosis of tumor cells. In this study, the genotoxic effects of oxidative hydrogen peroxide (H?O?) and alkylating agents N-methyl-N-nitrosourea (MNU) and 1,3-bis-(2-chloroethyl)-1-nitosourea (BCNU) agents were examined in two colon cell lines (HCT15 and CO115). DNA damage was assessed by the comet assay with and without lesion-specific repair enzymes. Genotoxic agents were used for induction of DNA damage in both cell lines. Protective effects of extracts of three Salvia species, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), against DNA damage induced by oxidative and alkylating agents were also determined. SO and SF protected against oxidative DNA damage in HCT15 cells. SO and SL decreased DNA damage induced by MNU in CO115 cells. In addition to chemopreventive effects of sage plant extracts, it was also important to know whether these plant extracts may interfere with alkylating agents such as BCNU used in cancer therapy, decreasing their efficacy. Our results showed that sage extracts tested and rosmarinic acid (RA), the main constituent, protected CO115 cells from DNA damage induced by BCNU. In HCT15 cells, only SF induced a reduction in BCNU-induced DNA damage. Sage water extracts and RA did not markedly change DNA repair protein expression in either cell line. Data showed that sage tea protected colon cells against oxidative and alkylating DNA damage and may also interfere with efficacy of alkylating agents used in cancer therapy. PMID:22788364

Ramos, Alice A; Pedro, Dalila; Collins, Andrew R; Pereira-Wilson, Cristina

2012-01-01

282

Comparison of four methods for extracting DNA from dried blood on filter paper for PCR targeting the mitochondrial Plasmodium genome  

PubMed Central

Background Few studies comparing multiple methods for DNA extraction from dried blood spots (DBS) on filter paper for PCR targeting the Plasmodium genome have been done. Methods Frequently-used methods for DNA extraction from DBS using Chelex-100, InstaGene Matrix, QIAamp DNA Mini Kit and TE buffer were compared on a dilution series of a standardized Plasmodium falciparum positive sample. The two DNA extraction methods resulting in the lowest limits of detection were compared by testing both on 31 P. falciparum positive samples collected under field conditions and stored for 4 years. Results The Chelex-100, InstaGene Matrix and QIAamp DNA Mini Kit methods performed similarly, resulting in the detection of 0.5 to 2 parasites per microliter (p/l). The same 13 clinical samples (13/31; 42%) were positive using both DNA extraction methods with the lowest limits of detection. Conclusions Simple and low-cost methods can be sensitive and useful in extracting DNA from DBS. Poor results on stored clinical DBS indicate that further studies on the impact of storage duration and conditions, and choice of filter paper should be performed. PMID:24907711

Strm, Gro E. A.; Tellevik, Marit G.; Hanevik, Kurt; Langeland, Nina; Blomberg, Bjrn

2014-01-01

283

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches.  

PubMed

A co-extraction protocol that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities is presented. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA- and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction. PMID:24929037

Gunnigle, Eoin; Ramond, Jean-Baptiste; Frossard, Aline; Seeley, Mary; Cowan, Don

2014-08-01

284

Effect of extracts from pine needle against oxidative DNA damage and apoptosis induced by hydroxyl radical via antioxidant activity  

Microsoft Academic Search

In this study, the protective effects of water extracts from pine needle (WEPN) against DNA damage and apoptosis induced by hydroxyl radical were investigated in non-cellular and cellular system. WEPN exhibited strong scavenging action on hydroxyl radical and intracellular ROS, and chelating action of Fe2+ ion. WEPN inhibited oxidative DNA damage by hydroxyl radical. Also, WEPN prevented the cells from

Jin Boo Jeong; Eul Won Seo; Hyung Jin Jeong

2009-01-01

285

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

Microsoft Academic Search

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

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

1982-01-01

286

Assessment of nematode biodiversity using DGGE of 18S rDNA following extraction of nematodes from soil  

Microsoft Academic Search

Soil nematodes are both taxonomically and functionally diverse, respond quickly to soil perturbation and have much potential as indicators of soil health. However, because of the perceived difficulty of identifying nematodes to species level morphologically, they are frequently neglected in soil ecological studies. Recently, extraction of soil DNA, amplification of 18S rDNA genes using nematode consensus primers and subsequent separation

Aude L. J. L. Foucher; Tom Bongers; Leslie R. Noble; Michael J. Wilson

2004-01-01

287

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae  

Microsoft Academic Search

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating

Yasuko Matsumoto; Tsuyoshi Nakano; Masafumi Yamamoto; Yuko Matsushima-Hibiya; Ken-Ichi Odagiri; Osamu Yata; Kotaro Koyama; Takashi Sugimura; Keiji Wakabayashi

2008-01-01

288

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

289

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

290

Quantitation of 35S promoter in maize DNA extracts from genetically modified organisms using real-time polymerase chain reaction, part 2: interlaboratory study.  

PubMed

The European Committee for Standardization (CEN) and the European Network of GMO Working Laboratories have proposed development of a modular strategy for stepwise validation of complex analytical techniques. When applied to the quantitation of genetically modified organisms (GMOs) in food products, the instrumental quantitation step of the technique is separately validated from the DNA extraction step to better control the sources of uncertainty and facilitate the validation of GMO-specific polymerase chain reaction (PCR) tests. This paper presents the results of an interlaboratory study on the quantitation step of the method standardized by CEN for the detection of a regulatory element commonly inserted in GMO maize-based foods. This is focused on the quantitation of P35S promoter through using the quantitative real-time PCR (QRT-PCR). Fifteen French laboratories participated in the interlaboratory study of the P35S quantitation operating procedure on DNA extract samples using either the thermal cycler ABI Prism 7700 (Applied Biosystems, Foster City, CA) or Light Cycler (Roche Diagnostics, Indianapolis, IN). Attention was focused on DNA extract samples used to calibrate the method and unknown extract samples. Data were processed according to the recommendations of ISO 5725 standard. Performance criteria, obtained using the robust algorithm, were compared to the classic data processing after rejection of outliers by the Cochran and Grubbs tests. Two laboratories were detected as outliers by the Grubbs test. The robust precision criteria gave values between the classical values estimated before and after rejection of the outliers. Using the robust method, the relative expanded uncertainty by the quantitation method is about 20% for a 1% Bt176 content, whereas it can reach 40% for a 0.1% Bt176. The performances of the quantitation assay are relevant to the application of the European regulation, which has an accepted tolerance interval of about +/-50%. These data were fitted to a power model (r2 = 0.96). Thanks to this model, it is possible to propose an estimation of uncertainty of the QRT-PCR quantitation step and an uncertainty budget depending on the analytical conditions. PMID:15859084

Feinberg, Max; Fernandez, Sophie; Cassard, Sylvanie; Bertheau, Yves

2005-01-01

291

C-C1-02: Data Extraction From Text, Step 1: Preparing Test for Machine Processing  

PubMed Central

Background: Natural language processing (NLP) uses software to assist in the extraction of information from clinical text, a process usually performed entirely by chart abstractors. Before NLP can be applied the text in question must be prepared for machine processing. In research settings this pre- processing work often involves several successive and related tasks, requiring substantial amounts of time and attention from people representing various types of clinical, scientific and technical expertise. Appreciating the tasks and participants involved in pre-processing clinical text can make the work more manageable, efficient, and effective. Methods: The information presented here comes from case study analyses of three small-scale projects involving preparation of clinical text (pathology reports, radiology reports, and progress notes) for processing by the Cancer Text Information Extraction System. Supplementing these experiences is information from anecdotal conversations with natural language processing experts. Results: Ten separate pre-processing tasks were identified: obtaining source feeds, assessing completeness, de-duplication, universe description, cleaning and formatting, de-identification, database loading, sampling, preparation of the NLP system input feed, and quality assurance. Nine types of expertise or task participants required for preprocessing were identified: IRB representative, source-system manager, network/dbase administrator, programmer, statistician, investigator, informaticist, clinical domain expert, and manual chart abstractor. Conclusions: Pre-processing clinical text is an important phase and potentially challenging aspect of extracting information from clinical text using NLP. Because researchers require accurate information about the larger universe of documents or patients represented by the sampled and processed text, pre-processing can present numerous challenges, the solutions to which draw on many areas of expertise in a multi-step and sometimes iterative process.

Carrell, David

2010-01-01

292

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

293

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

294

Rapid method for DNA extraction from the honey bee Apis mellifera and the parasitic bee mite Varroa destructor using lysis buffer and proteinase K.  

PubMed

We developed a rapid method for extraction of DNA from honey bees, Apis mellifera, and from the parasitic bee mite, Varroa destructor. The advantages include fast processing and low toxicity of the substances that are utilized. We used lysis buffer with nonionic detergents to lyse cell walls and proteinase K to digest proteins. We tested whole thorax, thoracic muscle mass, legs, and antennae from individual bees; the mites were processed whole (1 mite/sample). Each thorax was incubated whole, without cutting, because exocuticle color pigment darkened the extraction solution, interfering with PCR results. The procedure was performed with autoclaved equipment and laboratory gloves. For each sample, we used 100 L lysis buffer (2 mL stock solution of 0.5 M Tris/HCl, pH 8.5, 10 mL stock solution of 2 M KCl, 500 L solution of 1 M MgCl2, 2 mL NP40, and 27.6 g sucrose, completed to 200 mL with bidistilled water and autoclaved) and 2 L proteinase K (10 mg/mL in bidistilled water previously autoclaved, as proteinase K cannot be autoclaved). Tissues were incubated in the solutions for 1-2 h in a water bath (62-68 C) or overnight at 37 C. After incubation, the tissues were removed from the extraction solution (lysis buffer + proteinase K) and the solution heated to 92 C for 10 min, for proteinase K inactivation. Then, the solution with the extracted DNA was stored in a refrigerator (4-8 C) or a freezer (-20 C). This method does not require centrifugation or phenol/chloroform extraction. The reduced number of steps allowed us to sample many individuals/day. Whole mites and bee antennae were the most rapidly processed. All bee tissues gave the same quality DNA. This method, even using a single bee antenna or a single mite, was adequate for extraction and analysis of bee genomic and mitochondrial DNA and mite genomic DNA. PMID:24301746

Issa, M R C; Figueiredo, V L C; De Jong, D; Sakamoto, C H; Simes, Z L P

2013-01-01

295

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

296

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.  

PubMed Central

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt. Images PMID:8202366

Beyert, N; Reichenberger, S; Peters, M; Hartung, M; Gttlich, B; Goedecke, W; Vielmetter, W; Pfeiffer, P

1994-01-01

297

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

298

A novel reliable method of DNA extraction from olive oil suitable for molecular traceability.  

PubMed

Extra virgin olive oil production has a worldwide economic impact. The use of this brand, however, is of great concern to Institutions and private industries because of the increasing number of fraud and adulteration attempts to the market products. Here, we present a novel, reliable and not expensive method for extracting the DNA from commercial virgin and extra virgin olive oils. The DNA is stable overtime and amenable for molecular analyses; in fact, by carrying out simple sequence repeats (SSRs) markers analysis, we characterise the genetic profile of monovarietal olive oils. By comparing the oil-derived pattern with that of the corresponding tree, we can unambiguously identify four cultivars from Samnium, a region of Southern Italy, and distinguish them from reference and more widely used varieties. Through a parentage statistical analysis, we also identify the putative pollinators, establishing an unprecedented and powerful tool for olive oil traceability. PMID:25442596

Raieta, Katia; Muccillo, Livio; Colantuoni, Vittorio

2015-04-01

299

A novel approach on fluid dispensing for a DNA/RNA extraction chip package  

NASA Astrophysics Data System (ADS)

Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

2008-02-01

300

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

301

Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.  

PubMed

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C T ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.510(4) colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen. PMID:23460920

Podnecky, Nicole L; Elrod, Mindy G; Newton, Bruce R; Dauphin, Leslie A; Shi, Jianrong; Chawalchitiporn, Sutthinan; Baggett, Henry C; Hoffmaster, Alex R; Gee, Jay E

2013-01-01

302

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

303

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

PubMed

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

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

2014-01-01

304

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

PubMed Central

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

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

2014-01-01

305

Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.  

PubMed

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens. PMID:21159933

Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

2011-02-01

306

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae.  

PubMed

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of approximately 100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-02-19

307

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae  

PubMed Central

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ?100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-01-01

308

Determination of gold, indium, tellurium and thallium in the same sample digest of geological materials by atomic-absorption spectroscopy and two-step solvent extraction  

USGS Publications Warehouse

A rock, soil, or stream-sediment sample is decomposed with hydrofluoric acid, aqua regia, and hydrobromic acid-bromine solution. Gold, thallium, indium and tellurium are separated and concentrated from the sample digest by a two-step MIBK extraction at two concentrations of hydrobromic add. Gold and thallium are first extracted from 0.1M hydrobromic acid medium, then indium and tellurium are extracted from 3M hydrobromic acid in the presence of ascorbic acid to eliminate iron interference. The elements are then determined by flame atomic-absorption spectrophotometry. The two-step solvent extraction can also be used in conjunction with electrothermal atomic-absorption methods to lower the detection limits for all four metals in geological materials. ?? 1985.

Hubert, A.E.; Chao, T.T.

1985-01-01

309

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

310

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

311

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis  

PubMed Central

OBJECTIVES: The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2. INTRODUCTION: The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti?cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail. METHODS: HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0?4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis. RESULTS: Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro? apoptotic proteins P53, Bax and caspase?3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti?apoptotic protein Bcl?2. CONCLUSIONS: Chlorella vulgaris may have anti?cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase?3 proteins and through a reduction of Bcl?2 protein, which subsequently lead to increased DNA damage and apoptosis. PMID:21340229

Yusof, Yasmin Anum Mohd; Md. Saad, Suhana; Makpol, Suzana; Shamaan, Nor Aripin; Ngah, Wan Zurinah Wan

2010-01-01

312

The Mechanism of Specific Cleavage of Supercoiled DNA by Human DNA Topoisomerase I: The Effect of Ligand Structure on the Catalytic Step of Cleavage  

Microsoft Academic Search

Eukaryotic DNA topoisomerase I (Topo) regulates the topological state of cell DNA and plays an important part in replication, transcription, repair, and recombination. Factors affecting the specific recognition of topologically stressed DNA were analyzed on the basis of the thermodynamic and kinetic data on the TopoDNA interaction and the X-ray data on human Topo. A model was advanced for possible

D. V. Bugreev; V. N. Buneva; G. A. Nevinsky

2003-01-01

313

One-Step Analysis of DNA/Chitosan Complexes by Field-Flow Fractionation Reveals Particle Size and Free Chitosan Content  

E-print Network

One-Step Analysis of DNA/Chitosan Complexes by Field-Flow Fractionation Reveals Particle Size and Free Chitosan Content Pei Lian Ma, Michael D. Buschmann, and Franc¸oise M. Winnik*, Department with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV

Buschmann, Michael

314

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

315

Efficient extraction of viral DNA and viral RNA by the Chemagic viral DNA/RNA kit allows sensitive detection of cytomegalovirus, hepatitis B virus, and hepatitis G virus by PCR.  

PubMed

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available. PMID:14605182

Kleines, Michael; Schellenberg, Kirsten; Ritter, Klaus

2003-11-01

316

Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood.  

PubMed

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction. PMID:22238432

Germi, Raphale; Lupo, Julien; Semenova, Touyana; Larrat, Sylvie; Magnat, Nelly; Grossi, Laurence; Seigneurin, Jean-Marie; Morand, Patrice

2012-04-01

317

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

318

A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods.  

PubMed

The Metropolitan Police Service currently uses cotton swabs to retrieve DNA for forensic profiling. Recently, a new nylon flocked swab type has become available from Copan (MicroRheologics, Brescia, Italy) that it is claimed, offers increased sample recovery and release yields. If true, the flocked swab may have important applications in DNA evidence retrieval. This study examines the DNA retrieval capability of cotton and nylon flocked swabs when extracted using three common extraction platforms (QIAcube, BioRobot EZ1 and manually processed QIAamp DNA investigator kit). Results indicate that both swab types are capable of recovering high percentages of DNA (>50%); however, the extraction platform selected was shown to have a significant effect upon DNA retrieval. Across all experiments, the cotton swab combined with the spin-column extractions was shown to be most effective, with the nylon swab and BioRobot EZ1 combination being the least effective. These findings illustrate the importance of extraction method selection. PMID:22211626

Brownlow, Robert J; Dagnall, Kathryn E; Ames, Carole E

2012-05-01

319

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

320

Quantification and presence of human ancient DNA in burial place remains of Turkey using real time polymerase chain reaction  

Microsoft Academic Search

Archaeometry and forensic laboratories are increasingly confronted with problematic samples from the scene of samples, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR) inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. Genomic DNA was

Hasibe Cingilli Vural

2009-01-01

321

Mismatch-specific thymine DNA glycosylase and DNA polymerase. beta. mediate the correction of Gter dot T mispairs in nuclear extracts from human cells  

SciTech Connect

To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G{center dot}T mispairs, arising in DNA as a result of this process, should always be corrected to G{center dot}C pairs. The authors describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. They further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase {beta}. This finding substantiates the proposed role of this enzyme in short-patch DNA repair.

Wiebauer, K.; Jiricny, J. (Friedrich Miescher Institute, Basel (Switzerland))

1990-08-01

322

An integrated disposable device for DNA extraction and helicase dependent amplification  

PubMed Central

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium. PMID:20066496

Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y.

2010-01-01

323

An integrated disposable device for DNA extraction and helicase dependent amplification.  

PubMed

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium. PMID:20066496

Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y; Klapperich, Catherine M

2010-04-01

324

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.  

PubMed

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results. PMID:25124940

Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

2014-11-01

325

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

326

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 Central

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

2010-01-01

327

The optimization of essential oils supercritical CO2 extraction from Lavandula hybrida through static-dynamic steps procedure and semi-continuous technique using response surface method  

PubMed Central

Aim: The aim of this study was to examine and evaluate crucial variables in essential oils extraction process from Lavandula hybrida through static-dynamic and semi-continuous techniques using response surface method. Materials and Methods: Essential oil components were extracted from Lavandula hybrida (Lavandin) flowers using supercritical carbon dioxide via static-dynamic steps (SDS) procedure, and semi-continuous (SC) technique. Results: Using response surface method the optimum extraction yield (4.768%) was obtained via SDS at 108.7 bar, 48.5C, 120 min (static: 815), 24 min (dynamic: 83 min) in contrast to the 4.620% extraction yield for the SC at 111.6 bar, 49.2C, 14 min (static), 121.1 min (dynamic). Conclusion: The results indicated that a substantial reduction (81.56%) solvent usage (kg CO2/g oil) is observed in the SDS method versus the conventional SC method. PMID:25598636

Kamali, Hossein; Aminimoghadamfarouj, Noushin; Golmakani, Ebrahim; Nematollahi, Alireza

2015-01-01

328

Detection of Transient Bacteraemia following Dental Extractions by 16S rDNA Pyrosequencing: A Pilot Study  

PubMed Central

Objective The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing. Methods The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene. Results Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.41.7 bacterial families and 22.81.1 genera per sample. Conclusion The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied. PMID:23469240

Bentez-Pez, Alfonso; lvarez, Maximiliano; Belda-Ferre, Pedro; Rubido, Susana; Mira, Alex; Toms, Inmaculada

2013-01-01

329

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

330

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 ...

331

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

E-print Network

of California-Santa Barbara, Santa Barbara, CA 93106-3210, United States 1. Introduction Numerous substances can given Forensic Science International 228 (2013) 142­153 A R T I C L E I N F O Article history: Received were evaluated for their efficacy against PCR inhibitors co-extracted with DNA from 63 ancient salmon

Kemp, Brian M.

332

Extraction of the metagenomic DNA and assessment of the bacterial diversity from the petroleum-polluted sites.  

PubMed

The assessment of the microbial diversity of the entire community of a given habitat requires the extraction of the total environmental DNA. Metagenomic investigations of a petroleum-polluted habitat have its unique challenges. The specific methods were developed for the extraction of high-quality metagenome in good quantity from the petroleum-polluted saline and non-saline sites in Gujarat (India). The soil samples were washed to remove the toxic, hazardous organic pollutants which might interfere with the recovery of the metagenomic DNA. The metagenomic DNA extraction results were encouraging with the mechanical bead beating, soft lysis, and combination of both. The extracted DNA was assessed for its purity and yield followed by its application in the amplification of the 16S rRNA region. The amplicons were used for judging the molecular diversity by the denaturing gradient gel electrophoresis (DGGE). The microbial diversity was also analyzed statistically by calculating various diversity indices and principal component analysis (PCA). The results on the metagenomic diversity of the bacterial population among the three cohorts based on the culture-independent technique exhibited significant difference among the PAH sites and Okha-Madhi and Porbandar Madhavpur habitats. PMID:24869956

Akbari, Viral G; Pandya, Rupal D; Singh, Satya P

2014-10-01

333

Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis  

Microsoft Academic Search

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of

Richard A. Haugland; Nichole Brinkman; Stephen J. Vesper

2002-01-01

334

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

E-print Network

Extraction of Plasmid DNA The majority of the work done with plasmids in M. tuberculosis strains for studies in M. tuberculosis are shuttle plasmids that have two plasmid origins of replication: one for use to retrieve a plasmid from an M. tuberculosis transformant to verify the integrity of an area of interest

335

Preliminary study of two steps aqueous two-phase extraction combined with high performance liquid chromatography for saliva protein selective separation.  

PubMed

Saliva protein separation by one-step aqueous two-phase extraction (ATPE) in the system of 15% (mass ratio if not specified) PEG-4000/8% NaH2PO4 was presented. The proteins extracted from the top and bottom phases were separated and characterized by high performance liquid chromatography (HPLC) respectively. Under the optimized gradient elution procedure, more saliva protein peaks in the two phases were shown. Saliva protein peaks in 50 min gradient were divided into ten specific groups which were classified into six sections based on the protein distribution in the top and bottom phases. Then, the top or bottom phase was performed the second step ATPE. Possible reasons for protein partition and retention difference are discussed briefly. Results show that some proteins can be separated completely in one-step ATPE, while some can be separated in the following two-step ATPE. The combination of two steps ATPE and HPLC provides a new way to achieve selective separation. PMID:25269257

Zhao, Xinying; Qu, Feng; Qin, Hao; Luo, Aiqin

2014-06-01

336

Protective effect of water yam (Dioscorea alata L.) extract on the copper-driven fenton reaction and X-ray induced DNA damage in vitro.  

PubMed

The rhizome extract of Dioscorea has been shown to possess radical scavenging activity. In this study, the protective effect of water yam (Dioscorea alata L.) rhizome extract on calf thymus DNA and plasmid DNA strand breakage by the copper-driven Fenton reaction and X-irradiation was examined. The protective activity in vitro of four lyophilized extracts obtained from yam rhizomes: (1) aqueous extract (YAE); (2) 30% ethanolic extract (YEE); (3) aqueous extract boiled for 30 min (BYAE); and (4) 30% ethanolic extract boiled for 30 min (BYEE) were evaluated by ethidium bromide binding assay and DNA nicking assay. The YAE, YEE, and BYEE effectively inhibited the copper-driven Fenton reaction-induced damage of calf thymus DNA, while inhibition was less pronounced in the case of X-ray induced strand breakage of plasmid DNA. While BYAE potently inhibited X-ray induced strand breaks in plasmid pGL3 DNA, it failed to inhibit, and even greatly enhanced copper-H(2)O(2) induced damage of calf thymus DNA. The present results demonstrate strong copper chelating and weak hydroxyl radical scavenging activities in yam rhizome extracts, and these activities may vary depending on the procedures used in preparing the extract. PMID:15162369

Wang, Tsu-Shing; Liang, Shih-Jyue; Lii, Chong-Kuei; Liu, Sin-Yie

2004-04-01

337

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

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 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; Sez, Claudio A; Brown, Murray T; Bitonti, Maria Beatrice

2014-01-01

338

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; Sez, Claudio A.; Brown, Murray T.; Bitonti, Maria Beatrice

2014-01-01

339

Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis  

PubMed Central

Background In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies. Results In this study we conducted a systematic comparison of the DNA extraction methods used by the two major collaborative efforts: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol. Conclusions Whereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies. PMID:24949196

2014-01-01

340

Wheat bran biorefinery: an investigation on the starch derived glucose extraction accompanied by pre- and post-treatment steps.  

PubMed

Wheat bran, a side product of the milling industry, can be considered as a feedstock for biorefineries. Unlike other lignocellulosic feedstock, wheat bran contains a reasonable amount of starch, which is not of recalcitrant nature. Therefore, it can be extracted without a costly pretreatment process. The present work evaluates the extraction of starch derived glucose in relation to a wheat bran biorefinery. The purity of free glucose extracted quantitatively was 44%. The extract was concentrated by threefold via nanofiltration, thereby reaching a glucose concentration of 49 g/L. Hydrothermal treatment (180C - 20 min) of the starch-free bran did not induce the formation of hydroxymethylfurfural and levulinic acid. Interestingly, the furfural level increased compared to the process, in which bran was treated hydrothermally without a preceding starch extraction. By separation of water-extractables prior to enzymatic hydrolysis, the free glucose purity was increased to 58%, however the yield of glucose decreased to 61%. PMID:24835741

Tirpanalan, zge; Reisinger, Michael; Huber, Florian; Kneifel, Wolfgang; Novalin, Senad

2014-07-01

341

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

Microsoft Academic Search

BackgroundRetrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter

Adam Kotorashvili; Andrew Ramnauth; Christina Liu; Juan Lin; Kenny Ye; Ryung Kim; Rachel Hazan; Thomas Rohan; Susan Fineberg; Olivier Loudig

2012-01-01

342

Cloud point extraction with surfactant derivatization as an enrichment step prior to gas chromatographic or gas chromatography-mass spectrometric analysis.  

PubMed

Cloud point extraction (CPE) using Triton X-114 was successfully applied as an extractive preconcentration step prior to gas chromatographic-mass spectrometric analysis. No liquid chromatographic or back-extraction steps were required to remove the target analyte(s) from the surfactant-rich extractant phase. Instead a post-extraction derivatization step is employed in which the surfactant of the surfactant-rich phase is reacted with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) prior to injection into the gas chromatograph. Such derivatization of the Triton X-114 surfactant following CPE was found to provide improved chromatographic performance yielding a reasonable elution time window that is free of derivatized surfactant signals, reproducible analyte retention times, and quantitative results. Mixtures of polycyclic aromatic hydrocarbons (PAHs), herbicides, and profens were utilized to demonstrate the feasibility and performance of this approach. The retention times of six PAHs (acenaphthene, acenaphthylene, anthracene, biphenyl, dibenzofuran, and fluorene) were found to be very reproducible with relative standard deviations (RSDs) in the range of 0.5-0.8%. Quantitative gas chromatography-mass spectrometry (GC/MS) analysis of a herbicide test mixture (composed of alachlor, atrazine, butachlor, hexachlorocyclopentadiene, metolachlor, and simazine) following their CPE from spiked water samples yielded detection limits in the range of 6.6-97 ng/L (except for that of hexachlorocyclopentadiene which was 482 ng/L). The enrichment factors achieved for these herbicides ranged from 17 to 33. The recovery of the herbicides from spiked water samples ranged from 90 to 100% except for simazine and atrazine which were 50% and 74%, respectively. The BSFTA derivatization step can serve not only to derivatize the surfactant but also appropriate nonvolatile (or less volatile) analytes. An ibuprofen and flurbiprofen test mix was utilized to demonstrate this feature. The proposed protocol offers an attractive alternative means by which surfactant-mediated extractions can be utilized as an enrichment step prior to gas chromatographic or gas chromatographic-mass spectrometric analysis of analytes which should serve to expand the scope of CPE in gas chromatographic (GC) analysis. PMID:19621897

Takagai, Yoshitaka; Hinze, Willie L

2009-08-15

343

Protective effects of aqueous and ethanolic extracts of Portulaca oleracea L. aerial parts on H2O2-induced DNA damage in lymphocytes by comet assay.  

PubMed

The comet assay is a standard method for measuring DNA damage. In this study, the protective effects of ethanolic and aqueous extracts of Portulaca oleracea L. (P. oleracea) on human lymphocyte DNA lesions were evaluated with the comet assay. Lymphocytes were isolated from blood samples taken from healthy volunteers. Human lymphocytes were incubated in H(2)O(2) (50,100, and 200 ?M), aqueous extract (0.05, 0.1, 0.5, 1, and 2.5mg/ml), and ethanolic extracts (0.05, 0.1, 0.5, 1, and 2.5mg/ml) of P. oleraceae aerial parts alone with a combination of H(2)O(2) (100 ?M) with either 1 or 2.5mg/ml of both extracts at 4C for 30 minutes. The extent of DNA migration was measured using the alkaline single cell gel electrophoresis approach assay, and DNA damage was expressed as percentage tail DNA. We found that the aqueous extract of P. oleracea significantly inhibited DNA damage, while there was no effect of the ethanolic extract. These data suggest that the aqueous extract of P. oleracea can prevent oxidative DNA damage to human lymphocytes, which is likely due to antioxidant constituents in the extract. PMID:21981871

Behravan, Javad; Mosafa, Fatemeh; Soudmand, Negar; Taghiabadi, Elahe; Razavi, Bibi Marjan; Karimi, Gholamreza

2011-09-01

344

Ancient DNA extracted from Danish aurochs (Bos primigenius): genetic diversity and preservation.  

PubMed

We extracted DNA from 39 Danish aurochs specimens and successfully amplified and sequenced a 252 base pair long fragment of the multivariable region I of the mitochondrial control region from 11 specimens. The sequences from these specimens dated back to 9830-2865 14Cyr BP and represent the first study of genetic variation of Danish aurochs. In addition, for all specimens we address correlations between the ability to obtain DNA sequences and various parameters such as the age of the sample, the collagen content, the museum storage period, Danish geography and whether the specimens were found in an archeological or geological context. We find that aurochs from southern Scandinavia display a star-shaped population genetic structure, that is indicative of a local and relatively recent diversification from a few ancestral haplotypes that may have originated in the ancestral Western European population before migration northwards during the retreat of the glaciers. Scenarios suggesting several invasions of genetically distinct aurochs are not supported by these analyses. Rather, our results suggest that a single continuous migration northward occurred. Our findings also suggest, although with only limited support, that aurochs in Northwestern Europe underwent a population expansion beginning shortly after the retreat of the glacial ice from Denmark and had a stable population size until the population decline that must have occurred prior to extinction. The absence of haplotypes similar to modern domestic cattle in our aurochs suggests that introgression between these species must have been limited, if it occurred at all. We found that the successful recovery of genetic material for PCR amplification correlates with sample age and local geographic conditions. However, contrary to other studies, we found no significant correlation between length of time in museum storage or the type of the locality in which a specimen was discovered (archeological or geological) and amplification success. Finally, we found large variances in our estimates of collagen content preventing an evaluation of this as an indicator of preservation quality. PMID:22188739

Gravlund, Peter; Aaris-Srensen, Kim; Hofreiter, Michael; Meyer, Matthias; Bollback, Jonathan P; Noe-Nygaard, Nanna

2012-01-20

345

Genotoxic Evaluation of Mikania laevigata Extract on DNA Damage Caused by Acute Coal Dust Exposure  

SciTech Connect

We report data on the possible antigenotoxic activity of Mikania laevigata extract (MLE) after acute intratracheal instillation of coal dust using the comet assay in peripheral blood, bone marrow, and liver cells and the micronucleus test in peripheral blood of Wistar rats. The animals were pretreated for 2 weeks with saline solution (groups 1 and 2) or MLE (100 mg/kg) (groups 3 and 4). On day 15, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg), and gross mineral coal dust (3 mg/0.3 mL saline) (groups 2 and 4) or saline solution (0.3 mL) (groups 1 and 3) was administered directly in the lung by intratracheal administration. Fifteen days after coal dust or saline instillation, the animals were sacrificed, and the femur, liver, and peripheral blood were removed. The results showed a general increase in the DNA damage values at 8 hours for all treatment groups, probably related to surgical procedures that had stressed the animals. Also, liver cells from rats treated with coal dust, pretreated or not with MLE, showed statistically higher comet assay values compared to the control group at 14 days after exposure. These results could be expected because the liver metabolizes a variety of organic compounds to more polar by-products. On the other hand, the micronucleus assay results did not show significant differences among groups. Therefore, our data do not support the antimutagenic activity of M. laevigata as a modulator of DNA damage after acute coal dust instillation.

Freitas, T.P.; Heuser, V.D.; Tavares, P.; Leffa, D.D.; da Silva, G.A.; Citadini-Zanette, V.; Romao, P.R.T.; Pinho, R.A.; Streck, E.L.; Andrade,V.M. [University of Extremo Catarinense, Criciuma, SC (Brazil)

2009-06-15

346

Lipid extracted algae as a source for protein and reduced sugar: A step closer to the biorefinery.  

PubMed

The objective of this study was to investigate the feasibility of using lipid extracted algae (LEA) as a source for protein and reduced sugar, and the effects of various procedural treatments on their yields. LEA provided comparable yields of protein and reduced sugars to those from total algae. Oven drying provided highest yields of all products followed by freeze drying, while sun drying significantly lowered their yields. Effective cell disruption by microwave and autoclave increased the lipid yields from algae, but resulted in increased loss of other compounds with lipid extracting solvents lowering their yields during sequential extraction. Relatively inefficient cell disruption by ultrasonication and osmotic shock lowered the amount of cell protein lost to the lipid extracting solvents. These results highlight the complexity of concurrent extraction of all value added products from algae, and the need for proper selection of the processes to achieve the objectives of integrated biorefinery. PMID:25579230

Ansari, Faiz Ahmad; Shriwastav, Amritanshu; Gupta, Sanjay Kumar; Rawat, Ismail; Guldhe, Abhishek; Bux, Faizal

2015-03-01

347

Step-gate polysilicon nanowires field effect transistor compatible with CMOS technology for label-free DNA biosensor  

E-print Network

-free DNA biosensor G. Wenga, E. Jacques, A-C. Salaün, R. Rogel, L. Pichon and F. Geneste* Institut d in "Biosensors and Bioelectronics Volume 40, Issue 1 (2013) 141-146" DOI : 10.1016/j.bios.2012.07.001 #12;Polycrystalline silicon nanowire FET (poly-Si NW FET); Bio-sensor; DNA hybridization; Spacer formation technique

Paris-Sud XI, Université de

348

Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues  

PubMed Central

Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types. PMID:24765569

Waggoner, Jesse J.

2014-01-01

349

The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis.  

PubMed

Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed. PMID:11605030

Bielawski, K; Zaczek, A; Lisowska, U; Dybikowska, A; Kowalska, A; Falkiewicz, B

2001-11-01

350

Comparison of four DNA extraction methods for detecting Mycobacterium tuberculosis by real-time PCR and its clinical application in pulmonary tuberculosis  

PubMed Central

Objectives China is one of the countries with a high burden of Mycobacterium tuberculosis (MTB) infection. One challenge for the earlier diagnosis of tuberculosis is the DNA extraction of MTB. This study was to compare four MTB DNA extraction methods, and use the best one in the diagnosis of pulmonary tuberculosis. Methods A total of 43 serum and 94 plasma samples were collected from 124 clinical diagnosed pulmonary tuberculosis patients. Four different MTB DNA extraction methods, including phenol-chloroform method, Qiagen kit, Omega kit and magnetic bead method, were compared to determine which method displayed the highest sensitivity. A quantitative fluorescent PCR assay was also designed for the detection of MTB DNA. Results The highest DNA extraction efficiency (52.8%) and the best reproducibility (coefficient of variance =26.7%) were observed using the magnetic bead method. For 39 of the 124 (31.5%) pulmonary tuberculosis patients, MTB DNA was detected in their plasma or serum samples. Interestingly, 35.3% (12/34) of smear-negative cases were MTB DNA positive. Conclusions In conclusion, magnetic bead method is the best one for the DNA extraction of MTB. The detection of MTB DNA may provide valuable information for the diagnosis of acid-fast bacilli (AFB) negative pulmonary tuberculosis patients. PMID:23825755

Gu, Bing; Yan, Zihe; Wang, Peng; Pei, Hao; Xie, Weiping; Chen, Dan; Liu, Genyan

2013-01-01

351

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing.  

PubMed

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation. PMID:25483069

Lipka, Daniel B; Wang, Qi; Cabezas-Wallscheid, Nina; Klimmeck, Daniel; Weichenhan, Dieter; Herrmann, Carl; Lier, Amelie; Brocks, David; von Paleske, Lisa; Renders, Simon; Wnsche, Peer; Zeisberger, Petra; Gu, Lei; Haas, Simon; Essers, Marieke Ag; Brors, Benedikt; Eils, Roland; Trumpp, Andreas; Milsom, Michael D; Plass, Christoph

2014-11-15

352

Quantification of microcystis in a eutrophic lake by simple DNA extraction and SYBR green real-time PCR.  

PubMed

A simple extraction method and real-time PCR with SYBR-Green I were combined to monitor Microcystis 16S rRNA gene (16S rDNA) concentrations over a wide range. A Fast DNA SPIN Kit (MP Biomedicals) was used to extract rDNA quantitatively. Real-time PCR amplified Microcystis rDNA for quantification with the forward primer Micro229f, which was newly designed by us and was highly specific for Microcystis, and the reverse universal primer 342r. The method developed here can detect Microcystis at concentrations as low as 3 cells mL(-1). The rDNA concentration and cell count were highly correlated in the range from 1.210(4) to 1.110(6) copies mL(-1) and from 9.510(2) to 1.010(5) cells mL(-1), respectively, in a canal where Microcystis algal blooms occur annually. The Microcystis rDNA concentration in Lake Kasumigaura was measured by the application of our method to water samples collected monthly from April 2004 to March 2006. Microcystis was not detected by microscopy from January to June 2005, except in May, but our method detected 1.010(3) to 1.010(4) copies mL(-1) of Microcystis rDNA during this period. This result clearly showed that our method is useful for clarifying the annual fluctuation in Microcystis concentration, especially when concentrations are low. PMID:21558723

Tomioka, Noriko; Nagai, Takashi; Kawasaki, Tatsuya; Imai, Akio; Matsushige, Kazuo; Kohata, Kunio

2008-01-01

353

Uncaria tomentosa extracts protect human erythrocyte catalase against damage induced by 2,4-D-Na and its metabolites.  

PubMed

The effect of ethanolic and aqueous extracts from leaves and bark of Uncaria tomentosa was studied, with particular attention to catalase activity (CAT - EC. 1.11.1.6). We observed that all tested extracts, at a concentration of 250 ?g/mL were not toxic to erythrocyte catalase because they did not decreased its activity. Additionally, we investigated the protective effect of extracts on changes in CAT activity in the erythrocytes incubated with sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D-Na) and its metabolites i.e., 2,4-dichlorophenol (2,4-DCP) and catechol. Previous investigations showed that these chemicals decreased activity of erythrocyte catalase (Bukowska et al., 2000; Bukowska and Kowalska, 2004). The erythrocytes were divided into two portions. The first portion was incubated for 1 and 5h at 37C with 2,4-D-Na, 2,4-DCP and catechol, and second portion was preincubated with extracts for 10 min and then incubated with xenobiotics for 1 and 5h. CAT activity was measured in the first and second portion of the erythrocytes. We found a protective effect of the extracts from U. tomentosa on the activity of catalase incubated with xenobiotics studied. Probably, phenolic compounds contained in U. tomentosa scavenged free radicals, and therefore protected active center (containing -SH groups) of catalase. PMID:22426356

Bukowska, Bo?ena; Bors, Milena; Gulewicz, Krzysztof; Koter-Michalak, Maria

2012-06-01

354

Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence detection through comigration with a digest.  

PubMed

We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays. PMID:15532567

Xu, Feng; Jabasini, Mohammad; Zhu, Bingmei; Ying, Ling; Cui, Xuezhi; Arai, Akihiro; Baba, Yoshinobu

2004-10-01

355

Mitochondrial DNA extraction and sequencing of formalin-fixed archival snake tissue  

Microsoft Academic Search

Formalinized specimens used in mitochondrial DNA (mtDNA) studies have shown shortcomings with respect to efficacy of DNA isolation and subsequent PCR amplification. In order to pinpoint the source of some of the problems with formalinized tissues in reptiles, we compared the efficacy of isolation and amplification of mtDNA from two different snake species Micrurus fulvius and Lampropeltis triangulumthat differ in

MIKE FRIEDMAN; ROB DESALLE

2008-01-01

356

Theoretical Study on the Relationship between Rp-Phosphorothioation and Base-Step in S-DNA: Based on Energetic and Structural Analysis.  

PubMed

Phosphorothioation (PT), previously used in synthetic antisense drugs to arrest the transcription or translation process, is also a novel physiological modification in bacteria DNAs. In the previous study, we reported that Rp-phosphorothioation (Rp-PT) destabilizes B-type helix significantly, using a quantum-mechanics-based energy scoring function developed with a dinucleotide model ( Zhang et al. J. Phys. Chem. B , 2012 , 116 , 10639 - 10648 ). A consequent question surfaces in the field of the phosphorothioated DNA (S-DNA) research: does the endogenous chemical modification interact with the base sequence in the bacterial genomes, e.g., in terms of the most common structure of the B-type helix? In this work, we carried out further energetic analysis on the backbone relative energies calculated with the scoring function according to 16 groups of base-step classifications. Moreover, we conducted molecular dynamics simulations of the B-helical structure with the different base-pair steps, to investigate the detailed structural changes upon the O-/S-substitution. As a result, the Rp-PT modification definitively enhances the stiffness of the backbone and differentiates backbone stability as an interaction with base-steps. Furthermore, certain exceptional sequences such as GT and CC were highlighted in the structural analysis of the sulfur local contacts and relative orientation of double strands, indicating that Rp-PT can cross-talk with particular base-steps. The special effects between the phosphorothioation and base-step may be related to the conservative consensus observed highly frequently in bacterial genomes. PMID:25519472

Chen, Limeng; Wang, Xiao-Lei; Shi, Ting; Wu, Tingting; Deng, Zixin; Zhao, Yi-Lei

2015-01-15

357

Semiconservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template 1 1 Edited by M. Yaniv  

Microsoft Academic Search

We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [?-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with

D Braguglia; P Heun; P Pasero; B. P Duncker; S. M Gasser

1998-01-01

358

Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.  

PubMed

Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types. PMID:24623253

Pfeiffer, Petra; Odersky, Andrea; Goedecke, Wolfgang; Kuhfittig-Kulle, Steffi

2014-01-01

359

Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol.  

PubMed

In Present work, the main objective is to develop less time consuming protocol for genomic DNA isolation from leaves of Passiflora foetida. Optimized protocol is cost effective, as it avoided use of expensive liquid nitrogen. The important parameters of CTAB buffer composition such as Polyvinylpyrrolidone PVP40000 (without PVP, 1%, 2%, 3.5%, 4.0%, 4.5%, 5.0%), CTAB (w, 1%, 2%, 3%, 4%, 5%), water bath temperature (30C to 70C) and duration on water bath for half hr and one and half hr has been optimized. CTAB (2%), PVP (1%), water bath temperature (70%), duration on water bath (1 hr) has efficiently yielded DNA quality of 200-1782 ?g/0.5gm from leaf, stem, root, tendril and flower. However, 168 ?g - 1782 ?g of DNA has been obtained from 0.5 g of leaf of Passiflora foetida. Polyphenol contamination has been overcome using 5M NaCl and PVP. Acetate has been used for obtaining double-stranded DNA in stabilized form. Current DNA extraction protocol takes maximum of four hours for completion, which is many time savings. RAPD-PCR reaction parameters such as DNA concentration (100ng), Primer concentration (2 ?M), Dream Taq polymerase (2 U), annealing temperature (29C) and number of cycles for amplification of DNA has been optimized. Primer fragment Akansha 7 shows high polymorphism of 7 fragments ranges from 200bp - 2500 bp. Current optimized protocol of DNA isolation is specifically for Passiflora foetida, which can be used for downstream molecular techniques. PMID:25191636

Lade, Bipin Deochand; Patil, Anita Surendra; Paikrao, Hariprassad Madhukarrao

2014-01-01

360

Development of an Alu-based, Real-Time PCR Method for Quantitation of Human DNA in Forensic Samples  

Microsoft Academic Search

Running header: Human Real-Time PCR Quantitation ABSTRACT: Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming and labor intensive. We have previously described a method for quantitation of human DNA

Janice A. Nicklas; Eric Buel

361

Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.  

PubMed

In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

2014-05-01

362

Rapid and sensitive diagnosis of fungal keratitis with direct PCR without template DNA extraction.  

PubMed

This study was aimed at developing a direct PCR assay without template DNA extraction for the rapid and sensitive diagnosis of infectious keratitis. Eighty corneal scrapings from 67 consecutive patients with clinically suspected infectious keratitis were analysed prospectively. Direct PCR was performed with all scrapings, with specific primers for fungi, bacteria, herpes simplex virus-1 (HSV-1) and Acanthamoeba simultaneously. The results were compared with those obtained from culture, smear, and confocal microscopy. Discrepant results were resolved according to the therapeutic effects of the corresponding antimicrobial drugs. The lowest detection limit of direct PCR was ten copies of each pathogen. Sixty-six scrapings yielded positive results with direct PCR, giving a total positive detection rate of 82.5% (66/80). For 34 patients with high suspicion of fungal keratitis, the positive detection rate of direct PCR was 84.8% (39/46). This rate increased to 91.2% (31/34) when repeated scrapings were excluded, and was significantly higher than the rates obtained with culture (35.3%, 12/34) and smear (64.7%, 22/34) (p <0.001), and was also higher than the rate obtained with confocal microscopy (74.1%, 20/27). The sensitivities for the diagnosis of infectious keratitis with direct PCR and culture were 98.0% and 47.1% (p <0.001), whereas the specificities were 81.8% and 100%, respectively. The time required to complete the entire direct PCR procedure was only 3 h. The direct PCR assay is a rapid diagnostic technique with high sensitivity and specificity for infectious keratitis, and it is expected to have an impact on the diagnosis and treatment of infectious keratitis in the future. PMID:24471925

Zhao, G; Zhai, H; Yuan, Q; Sun, S; Liu, T; Xie, L

2014-10-01

363

Micro-Doppler Effect Analysis and Feature Extraction in ISAR Imaging With Stepped-Frequency Chirp Signals  

E-print Network

The micro-Doppler (m-D) effect induced by the rotating parts or vibrations of the target provides a new approach for target recognition. To obtain high range resolution for the extraction of the fine m-D signatures of an ...

Luo, Ying

364

Molecular identification of fine roots of trees from the Alps: reliable and fast DNA extraction and PCR-RFLP analyses of plastid DNA.  

PubMed

Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 x 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots. PMID:11555251

Brunner, I; Brodbeck, S; Bchler, U; Sperisen, C

2001-08-01

365

Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems.  

PubMed

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Cline; Ciron, Pierre Emmanuel; Godfroy, Anne; Cambon-Bonavita, Marie-Anne

2014-08-01

366

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems  

PubMed Central

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Cline; Ciron, Pierre Emmanuel; Godfroy, Anne

2014-01-01

367

A rapid semi automated method for DNA extraction from dried-blood spots: Application to the HLA-DR shared epitope analysis in rheumatoid arthritis  

Microsoft Academic Search

Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, DBS-miniMAG, that combines filter paper dried blood spots (DBS) with the NucliSens miniMAG semi-automated instrument (bioMrieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in

Alexandre Pachot; Vronique Barbalat; Hubert Marotte; Jennifer Diasparra; Aurore Gouraud; Bruno Mougin; Pierre Miossec

2007-01-01

368

Autism and the extraction of emotion from briefly presented facial expressions: stumbling at the first step of empathy.  

PubMed

Identification of other people's emotion from quickly presented stimuli, including facial expressions, is fundamental to many social processes, including rapid mimicry and empathy. This study examined extraction of valence from brief emotional expressions in adults with autism spectrum disorder (ASD), a condition characterized by impairments in understanding and sharing of emotions. Control participants were individuals with reading disability and typical individuals. Participants were shown images for durations in the range of microexpressions (15 ms and 30 ms), thus reducing the reliance on higher level cognitive skills. Participants detected whether (a) emotional faces were happy or angry, (b) neutral faces were male or female, and (c) neutral images were animals or objects. Individuals with ASD performed selectively worse on emotion extraction, with no group differences for gender or animal?object tasks. The emotion extraction deficit remains even when controlling for gender, verbal ability, and age and is not accounted for by speed-accuracy tradeoffs. The deficit in rapid emotional processing may contribute to ASD difficulties in mimicry, empathy, and related processes. The results highlight the role of rapid early emotion processing in adaptive social?emotional functioning. PMID:19102591

Clark, Tedra F; Winkielman, Piotr; McIntosh, Daniel N

2008-12-01

369

Successful isolation and PCR amplification of DNA from National Institute of Standards and Technology herbal dietary supplement standard reference material powders and extracts.  

PubMed

Twenty-four herbal dietary supplement powder and extract reference standards provided by the National Institute of Standards and Technology (NIST) were investigated using three different commercially available DNA extraction kits to evaluate DNA availability for downstream nucleotide-based applications. The material included samples of Camellia, Citrus, Ephedra, Ginkgo, Hypericum, Serenoa, And Vaccinium. Protocols from Qiagen, MoBio, and Phytopure were used to isolate and purify DNA from the NIST standards. The resulting DNA concentration was quantified using SYBR Green fluorometry. Each of the 24 samples yielded DNA, though the concentration of DNA from each approach was notably different. The Phytopure method consistently yielded more DNA. The average yield ratio was 22 : 3 : 1 (ng/microL; Phytopure : Qiagen : MoBio). Amplification of the internal transcribed spacer II region using PCR was ultimately successful in 22 of the 24 samples. Direct sequencing chromatograms of the amplified material suggested that most of the samples were comprised of mixtures. However, the sequencing chromatograms of 12 of the 24 samples were sufficient to confirm the identity of the target material. The successful extraction, amplification, and sequencing of DNA from these herbal dietary supplement extracts and powders supports a continued effort to explore nucleotide sequence-based tools for the authentication and identification of plants in dietary supplements. PMID:19844865

Cimino, Matthew T

2010-03-01

370

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry  

Microsoft Academic Search

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR

Dario De Medici; Luciana Croci; Elisabetta Delibato; Simona Di Pasquale; Emma Filetici; Laura Toti

2003-01-01

371

32 P-POSTLABELING AND HPLC SEPARATION OF DNA ADDUCTS FORMED BYDIESEL EXHAUST EXTRACTS IN VITRO AND IN MOUSE SKIN AND LUNG AFTERTOPICAL TREATMENT  

EPA Science Inventory

Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form PAH- and nitrated-PAH-DNA adducts in rodent skin and lung. he aim of this study was to characterize by "P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro...

372

Repair kinetics of acrolein and (E)-4-hydroxy-2-nonenalderived DNA adducts in human colon cell extracts  

PubMed Central

?-3 and ?-6 Polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate ?,?-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al., Biochemistry 43 (2004) 75147521). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA. PMID:24113140

Choudhury, Sujata; Dyba, Marcin; Pan, Jishen; Roy, Rabindra; Chung, Fung-Lung

2014-01-01

373

Repair kinetics of acrolein- and (E)-4-hydroxy-2-nonenal-derived DNA adducts in human colon cell extracts.  

PubMed

?-3 and ?-6 polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate ?,?-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al. [42]). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA. PMID:24113140

Choudhury, Sujata; Dyba, Marcin; Pan, Jishen; Roy, Rabindra; Chung, Fung-Lung

2013-01-01

374

Probing the rate-limiting step for intramolecular transfer of a transcription factor between specific sites on the same DNA molecule by (15)Nz-exchange NMR spectroscopy.  

PubMed

The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)Nz-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. The half-life for intramolecular transfer (0.5-1 s) is many orders of magnitude larger than the calculated transfer time (1-100 ?s) by sliding, leading us to conclude that the intramolecular transfer rates measured by z-exchange spectroscopy represent the rate-limiting step for a one-base-pair shift from the specific site to the immediately adjacent nonspecific site. At zero concentration of added salt, the intramolecular transfer rate constants between sites of opposing polarity are smaller than those between sites of the same polarity, suggesting that hopping/flipping may become rate-limiting at very low salt concentrations. PMID:25253516

Ryu, Kyoung-Seok; Tugarinov, Vitali; Clore, G Marius

2014-10-15

375

Comment on ''Phase-shift extraction and wave-front reconstruction in phase-shifting interferometry with arbitrary phase steps''  

NASA Astrophysics Data System (ADS)

We comment on the recent Letter by Cai et al. [Opt. Lett. 28, 1808 (2003)] in which an approach to phase-shifting interferometry with arbitrary phase steps was proposed. Cai et al. based their method of phase shifting on the idea that the intensities of the reference and object beams can be measured previously, which actually makes the whole posterior phase-shifting procedure absolutely unnecessary. Their method is also based on the statement that the phase of the Fresnel diffraction pattern of a test object if generally a spatially random distribution, which in most situations is wrong.

Ferrari, Jos A.; Garbusi, Eugenio

2004-06-01

376

Controlling DNA compaction with cationic amphiphiles for efficient delivery systems A step forward towards non-viral Gene Therapy  

NASA Astrophysics Data System (ADS)

The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and Lipofectamine TM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane).

Savarala, Sushma

377

Extraction of PCR-usable DNA from trees adapted to arid environment  

Microsoft Academic Search

Genetic conservation programs in arid natural repertoire rely on molecular methods for diversity assessments. Molecular characterization involves the use of high molecular weight genomic DNA as starting material. Obtaining intact genomic DNA of sufficiently high quality, readily amplifiable using PCR is a primary goal of all molecular genetic studies. The aim of our study was to devise a method for

G Sablok; P Gahlot; A. K. Gupta; K Pareek; N. S. Shekhawat

2009-01-01

378

Extraction of nuclear DNA from rhinoceros horn and characterization of DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros.  

PubMed

Rhinoceros horn is now worth more, per unit weight, than gold, diamonds, or cocaine. Rhinoceros horn has been used in traditional Asian medicine as a presumed cure for a wide range of ailments. Rhinoceros poaching in South Africa has, on average, more than doubled each year over the past 5 years with the rapid economic growth in east and southeast Asia being assumed to be the primary factor driving the increased demand for horn. Here we report on the characterization of methods for genomic DNA extraction from rhinoceros horn and on DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. The DNA profiling system described includes 22 short tandem repeat (STR), or microsatellite, markers and a gender marker (ZF1), which have been used previously in various studies on rhinoceros. Using a ? value of 0.1, a conservative estimate of random match probability in 5 white rhinoceros ranged from 1:7.3x10(6) to 1:3.0x10(8). Given that the total population of white rhinoceros is approximately 20,000 such random match probabilities indicate that the genotyping system described provides data which can be used for evidentiary purposes. Furthermore, the methods are appropriate for use in investigations involving trace amounts of rhinoceros horn and the matching of profiles obtained from seized rhinoceros horn with material collected from live animals or poached carcasses. PMID:23768315

Harper, Cindy K; Vermeulen, Gerhard J; Clarke, Amy B; de Wet, Jacobus I; Guthrie, Alan J

2013-07-01

379

One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold nanoparticles from Memecylon umbellatum  

PubMed Central

In this experiment, green-synthesized silver and gold nanoparticles were produced rapidly by treating silver and gold ions with an extract of Memecylon umbellatum leaf. The reaction process was simple and easy to handle, and was monitored using ultraviolet-visible spectroscopy. The effect of the phytochemicals present in M. umbellatum, including saponins, phenolic compounds, phytosterols, and quinones, on formation of stable silver and gold nanoparticles was investigated by Fourier-transform infrared spectroscopy. The morphology and crystalline phase of the nanoparticles were determined by transmission electron microscopy and energy-dispersive x-ray spectroscopy. The results indicate that the saponins, phytosterols, and phenolic compounds present in the plant extract play a major role in formation of silver and gold nanoparticles in their respective ions in solution. The characteristics of the nanoparticles formed suggest application of silver and gold nanoparticles as chemical sensors in the future. Given the simple and eco-friendly approach for synthesis, these nanoparticles could easily be commercialized for large-scale production. PMID:23569372

Arunachalam, Kantha D; Annamalai, Sathesh Kumar; Hari, Shanmugasundaram

2013-01-01

380

Improved protocol for high-quality co-extraction of DNA and RNA from rumen digesta.  

PubMed

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression. PMID:20680573

Popova, M; Martin, C; Morgavi, D P

2010-07-01

381

Comparative study on two-step concentrated acid hydrolysis for the extraction of sugars from lignocellulosic biomass.  

PubMed

Among all the feasible thermochemical conversion processes, concentrated acid hydrolysis has been applied to break the crystalline structure of cellulose efficiently and scale up for mass production as lignocellulosic biomass fractionation process. Process conditions are optimized by investigating the effect of decrystallization sulfuric acid concentration (65-80 wt%), hydrolysis temperature (80C and 100C), hydrolysis reaction time (during two hours), and biomass species (oak wood, pine wood, and empty fruit bunch (EFB) of palm oil) toward sugar recovery. At the optimum process condition, 78-96% sugars out of theoretically extractable sugars have been fractionated by concentrated sulfuric acid hydrolysis of the three different biomass species with 87-90 g/L sugar concentration in the hydrolyzate and highest recalcitrance of pine (softwood) was determined by the correlation of crystallinity index and sugar yield considering reaction severity. PMID:24859214

Wijaya, Yanuar Philip; Putra, Robertus Dhimas Dhewangga; Widyaya, Vania Tanda; Ha, Jeong-Myeong; Suh, Dong Jin; Kim, Chang Soo

2014-07-01

382

Applications of quantum mechanical/molecular mechanical methods to the chemical insertion step of DNA and RNA polymerization.  

PubMed

We review theoretical attempts to model the chemical insertion reactions of nucleoside triphosphates catalyzed by the nucleic acid polymerases using combined quantum mechanical/molecular mechanical methodology. Due to an existing excellent database of high-resolution X-ray crystal structures, the DNA polymerase ? system serves as a useful template for discussion and comparison. The convergence of structures of high-quality complexes and continued developments of theoretical techniques suggest a bright future for understanding the global features of nucleic acid polymerization. PMID:25458356

Perera, Lalith; Beard, William A; Pedersen, Lee G; Wilson, Samuel H

2014-01-01

383

Replication of M13 single-stranded viral DNA bearing single site-specific adducts by escherichia coli cell extracts: differential efficiency of translesion DNA synthesis for SOS-dependent and SOS-independent lesions.  

PubMed

In order to characterize mutagenic translesion DNA synthesis in UVM-induced Escherichia coli, we have developed a high-resolution DNA replication system based on E. coli cell extracts and M13 genomic DNA templates bearing mutagenic lesions. The assay is based on the conversion of M13 viral single-stranded DNA (ssDNA) bearing a single site-specific DNA lesion to the double-stranded replicative form (RF) DNA, and permits one to quantitatively measure the efficiency of translesion synthesis. Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion. In contrast, the efficiency of translesion synthesis across SOS-independent lesions such as O6-methylguanine and DNA uracil is around 90%, very close to the values obtained for control DNA templates. The efficiency of translesion synthesis across 3,N4-ethenocytosine and 1, N6-ethenoadenine is around 20%, a value that is similar to the in vivo efficiency deduced from the effect of the lesions on the survival of transfected M13 ssDNA. Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essentially similar results are obtained with extracts from polA- or polB-defective cells. The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions included in this study suggest that the assay may be suitable for modeling mutagenesis in an accessible in vitro environment. PMID:9235993

Wang, G; Rahman, M S; Humayun, M Z

1997-08-01

384

Comparison of three DNA extraction methods for feed products and four amplification methods for the 5'-junction fragment of Roundup Ready soybean.  

PubMed

Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients. PMID:22515503

Wang, Xiumin; Teng, Da; Tian, Fang; Guan, Qingfeng; Wang, Jianhua

2012-05-01

385

Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface  

PubMed Central

We present molecular threading, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers. PMID:23935923

Kemmish, Kent; Hamalainen, Mark; Bowell, Charlotte; Bleloch, Andrew; Klejwa, Nathan; Lehrach, Wolfgang; Schatz, Ken; Stark, Heather; Marblestone, Adam; Church, George; Own, Christopher S.; Andregg, William

2013-01-01

386

Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus  

PubMed Central

Background Host identification is an essential step in studies on the transmission dynamics of vector-borne disease. Nowadays, molecular tools allow the identification of vertebrate hosts to the species level. However, the proportion of successful identifications is variable and may be affected by the quality of the samples and the laboratory protocols. Here, the effect of two of these factors, namely the digestion status of mosquito blood meal and the DNA extraction procedure, on the success of host identification by amplification and sequencing of a fragment of the cytochrome oxidase 1 gene were tested. Methods Mosquitoes collected both outdoors and indoors during 2012 in southern Spain were identified to species level and their blood meal digestion status recorded using the Sella score, a visual estimation of the digestion status of mosquito blood meals. Each mosquito was assigned randomly to one of two DNA extraction procedures: the quick and cheap HotSHOT procedure or the QIAGEN DNeasy Blood and Tissue kit and their hosts identified by a molecular method. Results Three hundred and forty-seven blood-fed mosquitoes belonging to Anopheles atroparvus (n=171), Culex perexiguus (n=84), Culex pipiens (n=43), Culex theileri (n=39), Culex modestus (n=5), Ochlerotatus caspius (n=4), Culiseta sp. (n=1) were included in this study. Overall, hosts were identified from 234 blood meals compromising at least 25 species including mammals, birds and a single reptile. The success of host identification was lower in mosquitoes with an advanced stage of blood meal digestion and for blood meals extracted using the HotSHOT procedure. Conclusions The success of host identification decreases with the advanced stage of mosquito blood meal digestion, from 84.5% for recent blood meals to 25.0% for more digested ones. Using the QIAGEN kit, the identification success improved by 17.6%, with larger increases at more advanced stages of blood meal digestion. Availability of blood-fed females used to be very limited for studies of vector ecology, and these results may help to increase the efficiency of blood meal analyses. In addition, results obtained in this study clearly support that the potential malaria vector An. atroparvus feeds on animals located outdoors but use human-made shelters for resting after feeding. PMID:23517864

2013-01-01

387

Mitochondrial DNA extraction and sequencing of formalin-fixed archival snake tissue.  

PubMed

Formalinized specimens used in mitochondrial DNA (mtDNA) studies have shown shortcomings with respect to efficacy of DNA isolation and subsequent PCR amplification. In order to pinpoint the source of some of the problems with formalinized tissues in reptiles, we compared the efficacy of isolation and amplification of mtDNA from two different snake species Micrurus fulvius and Lampropeltis triangulum--that differ in composition of tissues typically sampled (intercostal muscle tissue) by researchers performing mtDNA analyses in snakes and other reptiles. This study shows clearly that the more muscle-fiber rich L. triangulum tissues yield higher quality mtDNA that is easier to manipulate with PCR than the myocyte depauperate Micrurus tissues. We suggest that curatorial practice in making available formalin preserved specimens in reptiles should focus on tissue type, most appropriately on muscle-rich tissues. We reiterate previous caution about minimizing amplicon size and maximizing controls for contamination when working with formalin-preserved reptile tissues. PMID:19489136

Friedman, Mike; DeSalle, Rob

2008-10-01

388

Transcription of synthetic DNA containing sequences with dyad symmetry by wheat-germ RNA polymerase II. Increased rates of product release in single-step addition reactions.  

PubMed

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II. PMID:1999201

Job, D; Job, C; de Mercoyrol, L; Shire, D

1991-02-14

389

DNA.  

ERIC Educational Resources Information Center

Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

Felsenfeld, Gary

1985-01-01

390

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes.  

PubMed

We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yel-low etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex-traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using proteinase K, and pre-cipitation of polysaccharides in the presence of a high salt concentra-tion. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calcu-lated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproduc-ible, and suitable for PCR-based markers. PMID:25501244

Ejaz, M; Gaisheng, Z; Na, N; Huiyan, Z; Qidi, Z; Qunzhu, W

2014-01-01

391

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids(,) (.)  

PubMed

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet() kit performed best based on average DNA (41.4ng) and mRNA (4.07ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. PMID:25284026

Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

2014-10-01

392

Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.  

PubMed

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Mnnedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpF?STR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. PMID:21609694

Stangegaard, Michael; Frslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

2011-04-01

393

Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study.  

PubMed

Anthocyanin extracts (AEs) from Ocimum tenuiflorum (leaf), Hibiscus rosa-sinensis (petal) and Hibiscus sabdariffa (calyx) were investigated and compared for in vitro antioxidant activity and DNA damage protective property. Total phenolic content (TPC) and total anthocyanin content (TAC) of the AEs were determined and the major anthocyanins were characterised. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical-scavenging activity, 2-deoxy-D-ribose degradation assay and lipid peroxidation assay. The protective property of the AEs was also examined against oxidative DNA damage by H2O2 and UV using pUC19 plasmid. All the AEs particularly those from O. tenuiflorum demonstrated efficient antioxidant activity and protected DNA from damage. Strong correlation between antioxidant capacity and TPC and TAC was observed. Significant correlation between antioxidant capacity and TPC and TAC ascertained that phenolics and anthocyanins were the major contributors of antioxidant activity. PMID:24730725

Sarkar, Biswatrish; Kumar, Dhananjay; Sasmal, Dinakar; Mukhopadhyay, Kunal

2014-01-01