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1

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

PubMed

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

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

2012-01-01

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).

Stephens, Janice; Leach, Jan

2011-01-01

3

Extracting DNA  

NSDL National Science Digital Library

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

Science Netlinks;

2002-03-28

4

Onion DNA Extraction  

NSDL National Science Digital Library

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

Hays, Lana

2009-01-01

5

DNA Extraction Virtual Lab  

NSDL National Science Digital Library

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

2006-01-01

6

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

PubMed

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

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

2011-10-15

7

Simplified buccal DNA extraction with FTA ® Elute Cards  

Microsoft Academic Search

DNA isolation is the initial step of most genetic studies, and ideally it should use a reliable and non-invasive method. Buccal samples are adequate for such purposes, being painless, easy to collect and a very reliable DNA source. FTA® Elute Cards are relatively new on the market and are designed for rapid blood DNA extraction, in which DNA is solubilized

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

2009-01-01

8

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).

9

Extraction, detection and persistence of extracellular DNA in forest litter microcosms  

Microsoft Academic Search

A DNA extraction method was developed that preferentially extracted extracellular DNA rather than intracellular DNA from forest litter. The method purposely avoided the use of harsh chemicals and physical disruption steps used in total DNA extraction to release DNA from cells. The detection limit of PCR, determined by spiking forest litter samples with a dilution series of Choristoneura fumiferana MNPVegt?\\/lacZ+

L. S England; M. L Vincent; J. T Trevors; S. B Holmes

2004-01-01

10

DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP  

PubMed Central

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

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

2009-01-01

11

How to Extract DNA From Anything Living  

NSDL National Science Digital Library

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

Utah, University O.

2008-01-01

12

DNA extraction from plants: The use of pectinase  

Microsoft Academic Search

Several earlier protocols for extracting plant DNA or RNA do not work well for a variety of plants because contaminating substances\\u000a coprecipitate with the nucleic acids and, thus, are present even at the last DNA-hydration step. While DNA extraction protocols\\u000a have been published in which pectinase is employed to break down these contaminating substances, here we present an alternative\\u000a modified

Steven H. Rogstad; Brian Keane; Carolyn Howes Keiffer; Fred Hebard; Paul Sisco

2001-01-01

13

Determination of an efficient and reliable method for DNA extraction from ticks  

Microsoft Academic Search

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing,

Taoufik Jamal; Laurence Vial; Renaud Maillard; Antonia Suau; Arnaud Le Menach; Henri-Jean Boulouis; Muriel Vayssier-Taussat

2004-01-01

14

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

15

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

16

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

17

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

PubMed

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

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

2008-08-01

18

A rapid and simple method for extracting yeast mitochondrial DNA  

Microsoft Academic Search

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

Ali Gargouri; M. Curie

1989-01-01

19

DNA Extraction and Quantitation for Forensic Analysts  

NSDL National Science Digital Library

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

2011-05-18

20

Investigation of DNA extraction from hair shafts  

Microsoft Academic Search

Human hair shafts can be important forensic evidence for identification, but DNA typing, even of mitochondrial DNA (mtDNA), presented certain difficulties. We describe three DNA extraction methods from hair shafts, such as the phenol\\/chloroform method, NaI treatment method, and silica-beads method. In order to make an investigation of mtDNA amplification rate and efficiency, the amplifications of the mtDNA control region

K Takayanagi; H Asamura; K Tsukada; M Ota; S Saito; H Fukushima

2003-01-01

21

Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes.  

PubMed

Different methods were evaluated to extract DNA from pooled nematodes belonging to Anisakis, Contracaecum, Pseudoterranova and Hysterothylacium genera isolated from edible fish. Pooled DNA extraction is the first and compulsory step to allow the identification of a large number of samples through high-throughput DNA sequencing with drastic time and cost reductions. PMID:24845469

Seesao, Y; Audebert, C; Verrez-Bagnis, V; Merlin, S; Jérôme, M; Viscogliosi, E; Dei-Cas, E; Aliouat-Denis, C M; Gay, M

2014-07-01

22

DNA Nanoarchitectures: Steps towards Biological Applications.  

PubMed

DNA's remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During recent decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile-based assembly process is a useful tool to construct large and simple structures; the DNA origami method is suitable for the production of smaller, more sophisticated and well-defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures in molecular and cellular biology. PMID:24953971

Tintoré, Maria; Eritja, Ramon; Fábrega, Carmen

2014-07-01

23

A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.  

PubMed

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values?0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed. PMID:23684993

Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

2013-08-01

24

A simplified universal genomic DNA extraction protocol suitable for PCR.  

PubMed

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

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

2011-01-01

25

Step-wise supercritical extraction of carbonaceous residua  

DOEpatents

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

Warzinski, R.P.

1986-05-15

26

Step-wise supercritical extraction of carbonaceous residua  

DOEpatents

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

Warzinski, Robert P. (Venetia, PA)

1987-01-01

27

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

Microsoft Academic Search

Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization

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

2011-01-01

28

An easily automated, closed-tube forensic DNA extraction procedure using a thermostable proteinase  

Microsoft Academic Search

Most standard procedures for extracting DNA from forensic substrates involve manipulations that expose the sample to potential contamination and which reduce yields. Furthermore, most methods require centrifugation and\\/or solvent extraction steps that render them difficult to automate. We describe a simple closed-tube DNA extraction procedure using a proteinase from the thermophilic Bacillus species EA1 that produces good DNA yields from

D. Moss; S.-A. Harbison; D. J. Saul

2003-01-01

29

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

PubMed Central

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

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

2013-01-01

30

Single-step Charge Transport through DNA over Long Distances  

PubMed Central

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

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

2011-01-01

31

MAGNETIC ON-CHIP DNA EXTRACTION IN A DROPLET BASED MICROSYSTEM  

Microsoft Academic Search

Magnetic droplet manipulation is a promising new approach towards the miniaturization of bioanalytical procedures. We present a system that combines droplet microfluidics and magnetic microparticles for the extraction and purification of DNA from µl-sized lysed cell samples. The DNA is detected on-chip via fluorescent microscopy or via an off-chip amplification step. We are able to extract and detect the DNA

U. Lehmann; C. Vandevyver; V. K. Parashar; M. A. M. Gijs

32

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

Microsoft Academic Search

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

Rafaela González-Montelongo; M. Gloria Lobo; Mónica González

2010-01-01

33

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

PubMed Central

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

Li, Yan; Araki, Hiroyuki

2013-01-01

34

Rapid methods to extract DNA and RNA from Cryptococcus neoformans.  

PubMed

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations. PMID:12702347

Bolano, A; Stinchi, S; Preziosi, R; Bistoni, F; Allegrucci, M; Baldelli, F; Martini, A; Cardinali, G

2001-12-01

35

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

PubMed Central

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

2014-01-01

36

Extraction of Mycobacterium tuberculosis DNA: a Question of Containment  

Microsoft Academic Search

DNA fingerprinting of Mycobacterium tuberculosis by IS6110 restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism, M. tuberculosis remained viable during this process. These data suggest that the extraction of M. tuberculosis DNA should be performed within containment until complete. The standard method employed for DNA

Wendy Somerville; Louise Thibert; Kevin Schwartzman; Marcel A. Behr

2005-01-01

37

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.

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

38

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

PubMed

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

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

2010-01-19

39

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

Microsoft Academic Search

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

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

2004-01-01

40

A rapid and efficient assay for extracting DNA from fungi  

USGS Publications Warehouse

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

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

2002-01-01

41

Viral DNA Packaging: One Step at a Time  

NASA Astrophysics Data System (ADS)

During its life-cycle the bacteriophage ?29 actively packages its dsDNA genome into a proteinacious capsid, compressing its genome to near crystalline densities against large electrostatic, elastic, and entropic forces. This remarkable process is accomplished by a nano-scale, molecular DNA pump - a complex assembly of three protein and nucleic acid rings which utilizes the free energy released in ATP hydrolysis to perform the mechanical work necessary to overcome these large energetic barriers. We have developed a single molecule optical tweezers assay which has allowed us to probe the detailed mechanism of this packaging motor. By following the rate of packaging of a single bacteriophage as the capsid is filled with genome and as a function of optically applied load, we find that the compression of the genome results in the build-up of an internal force, on the order of ˜ 55 pN, due to the compressed genome. The ability to work against such large forces makes the packaging motor one of the strongest known molecular motors. By titrating the concentration of ATP, ADP, and inorganic phosphate at different opposing load, we are able to determine features of the mechanochemistry of this motor - the coupling between the mechanical and chemical cycles. We find that force is generated not upon binding of ATP, but rather upon release of hydrolysis products. Finally, by improving the resolution of the optical tweezers assay, we are able to observe the discrete increments of DNA encapsidated each cycle of the packaging motor. We find that DNA is packaged in 10-bp increments preceded by the binding of multiple ATPs. The application of large external forces slows the packaging rate of the motor, revealing that the 10-bp steps are actually composed of four 2.5-bp steps which occur in rapid succession. These data show that the individual subunits of the pentameric ring-ATPase at the core of the packaging motor are highly coordinated, with the binding of ATP and the translocation of DNA temporally segregated into two distinct phases of the mechanochemical cycle of the entire ring. Because this ring-ATPase is a member of the ASCE superfamily of ATPases, these results may have implications for a broad and diverse family of cellular motors.

Bustamante, Carlos; Moffitt, Jeffrey R.

42

Comparison of DNA extraction methods for multiplex polymerase chain reaction  

Microsoft Academic Search

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

Triin Viltrop; Kaarel Krjutškov; Priit Palta; Andres Metspalu

2010-01-01

43

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 van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

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

2014-01-01

44

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

PubMed Central

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

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

2012-01-01

45

An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue  

PubMed Central

Background: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60–80%. Aims: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. Method: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. Results: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. Conclusion: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.

Coura, R; Prolla, J C; Meurer, L; Ashton-Prolla, P

2005-01-01

46

A fully automatable enzymatic method for DNA extraction from plant tissues  

PubMed Central

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

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

2005-01-01

47

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing.  

PubMed

Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA(@) SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae. PMID:22760785

Guo, Feng; Zhang, Tong

2013-05-01

48

Experimental evaluation of the Liu-Beveridge dinucleotide step model of DNA structure  

Microsoft Academic Search

Methods for predicting DNA curvature have many possible applications. Dinucleotide step models describe DNA shape by characterization of helical twist, deflection angles and the direction of deflection for nearest neighbor base pairs. Liu and Beveridge have extended previous applications of dinucleotide step models with the development and qualitative vali- dation of a predictive method for sequence-dependent DNA curvature (the LB

Philip R. Hardwidge; L. James Maher

2001-01-01

49

Feature Extraction From DNA Sequences by Multifractal Analysis.  

National Technical Information Service (NTIS)

This paper presents feature extraction and estimation of multifractal measures of DNA sequences using a multifractal methodology and demonstrates a new scheme for identifying biological functionality, using information contained within the DNA sequences. ...

H. Zhang W. Kinsner

2001-01-01

50

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

51

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

PubMed

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

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

2013-11-15

52

Rapid extraction of PCR-competent DNA from recalcitrant environmental samples.  

PubMed

Advances in sequencing technologies have made the investigation of microbial ecology and community dynamics more tractable. The critical first step in such analyses is the efficient and representative recovery of PCR-competent DNA from complex environmental samples. All extraction protocols contain inherent biases, meaning that choice of method involves compromise between various factors, including efficiency, yield, universality, and representative extraction. Here, details are given for a routine method used in our laboratory to extract DNA from soils, sediments, biofilms, roots, and fungi. PMID:24515357

Gillings, Michael R

2014-01-01

53

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

2012-01-01

54

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

PubMed

Sufficient lysis of soil or sediment microbes is a critical step for analyzing microbial community structures and for preparing metagenomic DNA libraries. The present study compared lysis methods for recovering archaeal, bacterial, actinomycete, and fungal DNAs from a mangrove sediment sample. PCR results showed that individual procedures using SDS, lysozyme, sonication, freeze-thaw, microwave, and vigorous shaking could extract archaeal or bacterial DNA but failed for actinomycetes or fungi cells. In comparison, an integrated lysis procedure using SDS, lysozyme, and vigorous shaking successfully obtained fungal DNA, and a combination of SDS, lysozyme, vigorous shaking, and microwave treatments recovered DNA from actinomycetes. Denaturing gradient gel electrophoresis (DGGE) results showed that although single lysis procedures can lyse bacterial DNA, all of them assessed the indigenous bacterial community structure with significant biases. The integrated lysis protocols described in the present study could be useful for extracting DNA from various types of sediments. PMID:21081284

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

2011-02-01

55

A comparison of DNA extraction methods using Petunia hybrida tissues.  

PubMed

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

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

2013-09-01

56

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

57

A simple Chelex protocol for DNA extraction from Anopheles spp.  

PubMed

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

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

2013-01-01

58

Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei  

Microsoft Academic Search

BACKGROUND: Methods for the extraction of DNA from filamentous fungi are frequently laborious and time consuming because most of the available protocols include maceration in liquid nitrogen after the mycelium has been grown in a liquid culture. This paper describes a new method to replace those steps, which involves the growth of the mycelium on cellophane disks overlaid on solid

Alexandre Cassago; Rodrigo Alexandre Panepucci; Ana Maria Tortella Baião; Flavio Henrique-Silva

2002-01-01

59

A Simplified Method for Mitochondrial DNA Extraction from Head Hair Shafts  

Microsoft Academic Search

DNA isolation from hair shafts can involve a number of steps, each of which adds time to the procedure and increases the risk of contamination. A simple alkaline digestion procedure that directly dissolves hairs was developed and compared with a widely used glass grinding\\/organic extraction method, using samples collected from 30 volunteers with varying population ancestries, hair colors, and hair

Elizabeth A. Graffy; David R. Foran

2005-01-01

60

The extraction of fungal DNA from multiple large soil samples  

Microsoft Academic Search

A commercial electric paint shaker was utilized to enable the simultaneous extraction of DNA from multiple large soil samples. A bead homogenization procedure was used to extract DNA from soil samples added to tubes. Soils infested with chlamydospores of the phytopathogenic fungus Cylindrocarpon destructans were used to evaluate the procedure. Glass beads and zirconium-oxide beads of various diameters were compared

R. D. Reeleder; B. B. Capell; L. D. Tomlinson; W. J. Hickey

2003-01-01

61

Comparison of four DNA extraction methods for forensic application  

Microsoft Academic Search

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

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

62

Sequential extraction and single-step cold-acid extraction: A feasibility study for use with freshwater-canal sediments  

Microsoft Academic Search

This investigation examines metal release from freshwater sediment using sequential extraction and single-step cold-acid leaching.\\u000a The concentrations of Cd, Cr, Cu, Fe, Ni, Pb and Zn released using a standard 3-step sequential extraction (Rauret et al., 1999) are compared to those released using a 0.5 M HCl; leach. The results show that the three sediments behave in very\\u000a different ways

Samantha R. Cook; Andrew Parker

2006-01-01

63

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

PubMed Central

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

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

64

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

PubMed

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

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

2003-03-01

65

Extraction of DNA from the plant Kalanchoë daigremontiana.  

PubMed

This protocol describes how to isolate genomic DNA from leaves and stems of Kalanchoë 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

Garcês, Helena; Sinha, Neelima

2009-10-01

66

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

Microsoft Academic Search

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

Hongmei Wang; Katrina J. Edwards

2009-01-01

67

Efficient DNA extraction from hair shafts  

Microsoft Academic Search

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

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

68

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

Microsoft Academic Search

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

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

2012-01-01

69

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

PubMed Central

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P < 0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The MasterPure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Fredricks, David N.; Smith, Caitlin; Meier, Amalia

2005-01-01

70

Comparison of methods of DNA extraction from stream sediments  

SciTech Connect

In Upper Three Runs Creek (Aiken, SC) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments. and those in which cells are removed from sediments prior to lysis. DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields. However, if a Southern blot is to be performed, the Tsai method is recommended because of the high degree of DNA fragmentation observed with the other methods.

Leff, L.G.; Dana, J.R.; Shimkets, L.J. [Univ. of Georgia, Athens, GA (United States); Vaun McArthur, J. [Savannah River Ecology Lab., Aiken, SC (United States)

1995-03-01

71

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

PubMed Central

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

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

2012-01-01

72

Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk.  

PubMed

Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment. PMID:16357279

Cremonesi, P; Castiglioni, B; Malferrari, G; Biunno, I; Vimercati, C; Moroni, P; Morandi, S; Luzzana, M

2006-01-01

73

Preliminary validation of Prepfiler Express™ Extraction kit in AutoMate Express DNA Extraction System  

Microsoft Academic Search

A variety of challenging biological samples, including blood stains, saliva, semen, hair, bones, finger nails, among others, are often a part of our casework investigation. In this study, semen, blood samples and saliva swabs were extracted by several methods in order to optimize and validate the Prepfiler Express™ Extraction kit and the AutoMate Express DNA Extraction System. Results obtained with

F. Balsa; V. Bogas; P. Cunha; P. Brito; A. Serra; V. Lopes; M. Carvalho; L. Andrade; A. M. Bento; M. São Bento; F. Corte-Real; M. J. Anjos

74

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

Microsoft Academic Search

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

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

75

Extraction of high quality DNA from seized Moroccan cannabis resin (Hashish).  

PubMed

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

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

2013-01-01

76

The extraction and purification of microbial DNA from sediments  

Microsoft Academic Search

Summary A new method for the isolation of intracellular and extracellular DNA from a range of sediment types has been developed. This 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 gradient centrifugation and\\/or hydroxyapatite chromatography. Yields of 26 ~g intracellular DNA and 1

Andrew Ogram; Gary S. Sayler; Tamar Barkay

1987-01-01

77

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques  

Microsoft Academic Search

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were

Hong Yang; Edward M. Golenberg; Jeheskel Shoshani

1997-01-01

78

Comparison of DNA extraction methods for identification of human remains  

Microsoft Academic Search

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

Felicity Pagan; Cindy Lim; Mojca Keglovic; Dennis McNevin

2011-01-01

79

Comparison of DNA extraction methods for identification of human remains  

Microsoft Academic Search

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

Felicity Pagan; Cindy Lim; Mojca Keglovic; Dennis McNevin

2012-01-01

80

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.

2014-01-01

81

DNA extraction conditions from Porphyra perforata using LiCl  

Microsoft Academic Search

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% ß-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts

Yong-Ki Hong; Sang-Dal Kim; Miriam Polne-Fuller; Aharon Gibor

1995-01-01

82

Method for the extraction of high quantity and quality cell-free DNA from amniotic fluid  

PubMed Central

Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are promising biomarkers with various promising clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNA. Further improvements to the original protocol for the extraction of cffDNA from AF supernatant resulted in statistically significant higher yields of high quality cffDNA, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g. comparative genomic hybridization [CGH] micro arrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples as compared to the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNA from up to ten samples in less than three hours. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g. to reduce potential contamination). The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material, and furthermore, will allow for exploration of widely unknown genetic, pathophysiological and kinetic issues of cell-free fetal DNA in amniotic fluid.

Lapaire, Olav; Johnson, Kirby L.; Bianchi, Diana W.

2009-01-01

83

New DNA Extraction Methods for Casework Evidence  

Microsoft Academic Search

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

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

84

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

PubMed

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

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

2012-09-01

85

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

86

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 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.

Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

2013-01-01

87

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

PubMed

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

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

2007-07-01

88

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

PubMed

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

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

2011-03-01

89

[Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].  

PubMed

This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated. PMID:21063995

P?nar, Ahmet; Akyön, Yakut; Alp, Alpaslan; Ergüven, Sibel

2010-07-01

90

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

PubMed Central

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

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

2012-01-01

91

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.

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

2013-01-01

92

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

PubMed

Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples. PMID:21531187

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

2012-03-01

93

DNA extraction and fingerprinting of commercial rice cereal products  

Microsoft Academic Search

DNA was extracted from commercial rice cereal products using modified conventional methods (CTAB, SDS and a commercial kit) in large fragments (>3kb) and with relatively high yields (1.4–10.7?g DNA per g of sample) and was used as template for the amplification of a single copy rice gene (i.e. MIPS) fragment (ca. 850bp) and microsatellite DNAs (ca. 120–400bp). The cereal products

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

2006-01-01

94

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

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

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

2012-05-01

95

Single Fish Egg DNA Extraction for PCR Amplification  

Microsoft Academic Search

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative\\u000a DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction\\u000a from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and

Futoshi Aranishi

2006-01-01

96

DNA Extraction from Museum Specimens of Parasitic Hymenoptera  

PubMed Central

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

Andersen, Jeremy C.; Mills, Nicholas J.

2012-01-01

97

Comparisons of Three Automated Systems for Genomic DNA Extraction in a Clinical Diagnostic Laboratory  

PubMed Central

Purpose The extraction of nucleic acid is initially a limiting step for successful molecular-based diagnostic workup. This study aims to compare the effectiveness of three automated DNA extraction systems for clinical laboratory use. Materials and Methods Venous blood samples from 22 healthy volunteers were analyzed using QIAamp® Blood Mini Kit (Qiagen), MagNA Pure LC Nucleic Acid Isolation Kit I (Roche), and Magtration-Magnazorb DNA common kit-200N (PSS). The concentration of extracted DNAs was measured by NanoDrop ND-1000 (PeqLab). Also, extracted DNAs were confirmed by applying in direct agarose gel electrophoresis and were amplified by polymerase chain reaction (PCR) for human beta-globin gene. Results The corrected concentrations of extracted DNAs were 25.42 ± 8.82 ng/µL (13.49-52.85 ng/µL) by QIAamp® Blood Mini Kit (Qiagen), and 22.65 ± 14.49 ng/µL (19.18-93.39 ng/µL) by MagNA Pure LC Nucleic Acid Isolation Kit I, and 22.35 ± 6.47 ng/µL (12.57-35.08 ng/µL) by Magtration-Magnazorb DNA common kit-200N (PSS). No statistically significant difference was noticed among the three commercial kits (p > 0.05). Only the mean value of DNA purity through PSS was slightly lower than others. All the extracted DNAs were successfully identified in direct agarose gel electrophoresis. And all the product of beta-globin gene PCR showed a reproducible pattern of bands. Conclusion The effectiveness of the three automated extraction systems is of an equivalent level and good enough to produce reasonable results. Each laboratory could select the automated system according to its clinical and laboratory conditions.

Lee, Jong-Han; Park, Yongjung; Choi, Jong Rak; Lee, Eun Kyung

2010-01-01

98

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

99

Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2.  

PubMed

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity. PMID:9442103

Hua, X H; Newport, J

1998-01-26

100

Microfluidic DNA extraction using a patterned aluminum oxide membrane  

Microsoft Academic Search

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.

Jungkyu Kim; Bruce K. Gale

2006-01-01

101

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.

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

2007-01-01

102

Regulation of the initiation step of DNA replication by cyclin-dependent kinases  

Microsoft Academic Search

Cyclin-dependent kinases (CDKs) play a central role in the regulation of cell cycle progression in eukaryotes. The onset of\\u000a S phase, the initiation of chromosomal DNA replication, is a major cell cycle event that is regulated by CDKs. Eukaryotic\\u000a chromosomal DNA replication is highly regulated and occurs as a two-step reaction. The first reaction, known as licensing,\\u000a is essential for

Seiji Tanaka; Hiroyuki Araki

2010-01-01

103

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

PubMed

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

Hawash, Yousry

2014-06-01

104

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR  

PubMed Central

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

2014-01-01

105

One-step purification of metallothionein extracted from two different sources.  

PubMed

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein. PMID:15899374

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

2005-06-25

106

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts.  

PubMed

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA. PMID:22391207

Kumar, Sanjay; Yoo, Hae Yong; Kumagai, Akiko; Shevchenko, Anna; Shevchenko, Andrej; Dunphy, William G

2012-03-15

107

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

ERIC Educational Resources Information Center

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

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

2005-01-01

108

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

Microsoft Academic Search

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

Muralidhar L Hegde; Tapas K Hazra; Sankar Mitra

2008-01-01

109

PCR-based typing of DNA extracted from cigarette butts.  

PubMed

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 scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts. PMID:1931740

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

1991-01-01

110

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

PubMed

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

Springer, Nathan M

2010-11-01

111

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

PubMed

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

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

2004-01-01

112

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

113

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

Microsoft Academic Search

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

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

2007-01-01

114

An Evaluation of Techniques for the Extraction and Amplification of DNA from Naturally Shed Hairs  

Microsoft Academic Search

Hair can be a valuable source of DNA for the nonin- vasive study of human and nonhuman populations. However, hairs contain extremely small quantities of DNA, making the method used to extract the DNA of paramount importance. This study compares the ef- fectiveness of 4 different methods of DNA extraction from shed chimpanzee hair, as measured by the ability to

Linda Vigilant

2003-01-01

115

An improved method for genomic DNA extraction from cyanobacteria  

Microsoft Academic Search

We present an improved method for genomic DNA extraction from cyanobacteria by updating the earlier method from our group\\u000a (Sinha et al. 2001) that does not require lysozyme treatment or sonication to lyse the cells. This method use lysis buffer to lyse the cells\\u000a and also skips the initial treatments to remove the exopolysaccharides or to break the clumps. To

Shailendra P. SinghRajesh; Rajesh P. Rastogi; Donat-P. Häder; Rajeshwar P. Sinha

2011-01-01

116

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

Microsoft Academic Search

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

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

2010-01-01

117

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

PubMed Central

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

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

2011-01-01

118

DNA Replication of the Parvovirus Kilham Rat Virus. I. Characterization of Intracellular Forms of Viral DNA Extracted by Guanidine Hydrochloride.  

National Technical Information Service (NTIS)

The following experimental procedures are described: infection of rat kidney cell cultures with Kilham rat virus (KRV); labeling and extraction of DNA; sucrose density gradient sedimentation; digestion by S sub 1 nuclease; and chromatography of DNA on ben...

A. T. Li G. Lavelle R. W. Tennant

1977-01-01

119

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

PubMed Central

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

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

2011-01-01

120

Presence of opportunistic oil-degrading microorganisms operating at the initial steps of oil extraction and handling.  

PubMed

Hydrocarbon-degrading microorganisms from natural environments have been isolated and identified using culture-dependent or molecular techniques. However, there has been little research into the occurrence of microorganisms incorporated into crude oil in the initial steps of extraction and handling, which can reduce the quality of stored petroleum. In the present study, a packed-column reactor filled with autoclaved perlite soaked with crude oil was subjected to a continuous flow of sterile medium in order to determine the presence of potential hydrocarbon degraders. Microorganisms developed on the surface of the perlite within a period of 73 days. DNA was extracted from the biofilm and then PCR-amplified using 16S rRNA bacterial and archaeal primers and 18S rRNA eukaryotic primers. No amplification was obtained using archaeal primers. However, denaturing gradient gel electrophoresis (DGGE) revealed the presence of unique bands indicating bacterial and eukaryotic amplification. Excision of these bands, sequencing, and subsequent BLAST search showed that they corresponded to Bacillus sp. and Aspergillus versicolor. The fungus was later isolated from intact perlite in agar plates. A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp. Analysis of the petroleum components by gas chromatography showed that there n-alkanes, aromatic hydrocarbons, and carbazoles were degraded. PMID:16835842

Sánchez, Olga; Ferrera, Isabel; Vigués, Núria; Garcia de Oteyza, Tirso; Grimalt, Juan O; Mas, Jordi

2006-06-01

121

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

PubMed Central

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

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

2002-01-01

122

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

PubMed

The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification. PMID:15062955

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

2004-05-10

123

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

PubMed

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

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

2013-06-01

124

Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure.  

PubMed

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

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

2006-01-01

125

DNA extraction and analysis from processed coffee beans.  

PubMed

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

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

2005-11-01

126

Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts.  

PubMed

Methods of rapidly extracting chromosomal DNA from human pathogenic yeasts were used in mitochondrial DNA (mtDNA) studies. This paper is concerned with rapid and reliable methods of extracting mtDNA for sequence analysis for species or strain identification, and epidemiological study of medically important fungi and fungal infections. To determine the optimal method of mtDNA extraction without isolating mitochondria, we examined three commonly used methods: 1). boiling, 2). glass bead disruption, and 3). a commercially available kit. We assessed the amount and quality of DNAs using a spectrophotometer and specific polymerase chain reaction (PCR). The DNA yield depended on the extraction method used and the yeast species. An adequate amount of mtDNA was obtained with both glass beads and a commercially available kit to amplify the mitochondrial gene using PCR without isolating the mitochondria. These techniques are convenient for extracting DNA from a variety of small-scale samples. PMID:12403909

Yamada, Yohko; Makimura, Koichi; Merhendi, Hossain; Ueda, Kumiko; Nishiyama, Yayoi; Yamaguchi, Hideyo; Osumi, Masako

2002-08-01

127

One step behind to step ahead - femoral approach to stabilize and to extract functional pacing lead to regain venous access  

PubMed Central

Transvenous lead extraction can be a method to regain venous access. We present the case of a man, aged 67, with indications to upgrade an ICD to a resynchronization therapy device. Since innominate vein occlusion was diagnosed and extraction of an abandoned ventricular pacing lead did not provide lumen regain, a functional atrial lead was extracted with the femoral approach to stabilization and venous access was regained. Asymptomatic vein wall damage but no other complications were recorded. The simultaneous application of different techniques to regain venous access may allow success of the final procedure in system upgrading.

Maciag, Aleksander; Syska, Pawel; Kusmierski, Krzysztof; Broy, Beata

2013-01-01

128

One step behind to step ahead - femoral approach to stabilize and to extract functional pacing lead to regain venous access.  

PubMed

Transvenous lead extraction can be a method to regain venous access. We present the case of a man, aged 67, with indications to upgrade an ICD to a resynchronization therapy device. Since innominate vein occlusion was diagnosed and extraction of an abandoned ventricular pacing lead did not provide lumen regain, a functional atrial lead was extracted with the femoral approach to stabilization and venous access was regained. Asymptomatic vein wall damage but no other complications were recorded. The simultaneous application of different techniques to regain venous access may allow success of the final procedure in system upgrading. PMID:24570742

Maci?g, Aleksander; Syska, Pawe?; Ku?mierski, Krzysztof; Broy, Beata; Sterli?ski, Maciej

2013-01-01

129

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

PubMed

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

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

2012-09-01

130

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

PubMed

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

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

2012-01-01

131

A Novel Microfluidic Platform for Continuous DNA Extraction and Purification using Laminar Flow Magnetophoresis  

Microsoft Academic Search

We present a novel microfluidic platform using laminar-flow magnetophoresis for combined continuous extraction and purification of DNA. All essential unit operations (DNA binding, sample washing and DNA elution) are integrated on one single chip. The key function is the motion of magnetic beads given by the interplay of laminar flow and time-varying magnetic field. The time for extraction was 1

M. Karle; J. Miwa; G. Roth; R. Zengerle; F. von Stetten

2009-01-01

132

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 student’s t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

2014-01-01

133

Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA.  

PubMed Central

The ATP concentrations allowing transcription of both heavy- and light-strand of human mtDNA in a HeLa cell mitochondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p[NH]ppA), an ATP analog with a nonhydrolyzable beta-gamma bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous ATP. Evidence was obtained indicating that p[NH]ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process. Images

Narasimhan, N; Attardi, G

1987-01-01

134

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

PubMed Central

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

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

2012-01-01

135

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

Microsoft Academic Search

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

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

1994-01-01

136

One-step synthesis of highly biocompatible multi-shaped gold nanostructures with fruit extract.  

PubMed

In this study, the authors demonstrate the synthesis of various gold nanostructures through a one-step, green and complete bio-modulation approach. Nanoparticles were successfully synthesised by the addition of gold aqueous solution to fruit extracts, including orange, papaya, peach or lemon. The particles were of various shapes and sizes with high abundance, such as sphere, marigold, triangle and hexagon. The biocompatibility of the presented gold nanostructures was examined; haemolysis tests revealed a non-toxicity result in blood cell uptake of such gold nanostructures. This study opens the exciting possibility of synthesising various multi-shaped nanoparticles through a simple and green approach, as well as paving the way for future bio-applications. PMID:21495781

Tai, Y; Tran, N T T; Tsai, Y-C; Fang, J-Y; Chang, L-W

2011-06-01

137

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 50°C 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.

Pang, Ho-ming; Yeung, Edward S.

2000-01-01

138

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.  

PubMed

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpF?STR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples. PMID:23254459

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

139

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis  

Microsoft Academic Search

Purpose of work  A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals\\u000a is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with\\u000a oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities\\u000a of DNA are

Lily X. Zelaya-MolinaMaria; Maria A. Ortega; Anne E. Dorrance

2011-01-01

140

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

PubMed

It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples. PMID:22629615

Zheng, Lu; Gao, Naiyun; Deng, Yang

2012-01-01

141

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

PubMed Central

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

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

1994-01-01

142

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

PubMed Central

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

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

143

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

Microsoft Academic Search

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

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

2001-01-01

144

Computational delineation of the catalytic step of a high-fidelity DNA polymerase  

PubMed Central

The Bacillus fragment, belonging to a class of high-fidelity polymerases, demonstrates high processivity (adding ?115 bases per DNA binding event) and exceptional accuracy (1 error in 106 nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two-metal-ion mechanism, and requiring the proton on the terminal primer O3? to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of ?12 kcal/mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.

Venkatramani, Ravindra; Radhakrishnan, Ravi

2010-01-01

145

A simple DNA extraction method for PCR amplification from dry seeds of Brassica napus.  

PubMed

A simple and reliable DNA extraction method for dry seeds of Brassica napus has been developed in our laboratory. The NaCl and PVP were used to remove polysaccharides and polyphenols during DNA purification. The oil and proteins of dry seeds were removed only through centrifugation in this method. The RAPD amplification patterns have no obviously difference between the DNA extracted from dry seeds and fresh leaves extracted with control method. The good results of SSR molecular markers on the DNA of dry seeds of another 12 B. napus indicating that the DNA extracted from dry seeds was freedom from common contaminating compounds. In conclusion, this method could be widely used in DNA extraction from dry seeds of B. napus. PMID:19070063

Maoteng, Li; Jianmin, Liu; Zhangyi; Pei, Wang; Lu, Gan; Longjiang, Yu

2007-04-01

146

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

147

An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction.  

PubMed

Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb. PMID:21221926

Liu, Juan; Li, Jingquan; Feng, Li; Cao, Hui; Cui, Zhongli

2010-12-01

148

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.

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

2013-01-01

149

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

PubMed

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

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

2014-05-01

150

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...

151

A simple method for DNA extraction from sporophyte in the brown alga Laminaria japonica  

Microsoft Academic Search

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA

Gaoge Wang; Yuhui Li; Peng Xia; Delin Duan

2005-01-01

152

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

Microsoft Academic Search

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

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

1999-01-01

153

Specific DNA extraction through fluid channels with immobilization of layered double hydroxides on polycarbonate surface  

Microsoft Academic Search

The purpose of this study was to immobilize inorganic layered double hydroxides (LDHs) on the polycarbonate (PC) substrate as the media to extract the specific DNA molecules through fluidic system and to enhance the extraction efficiency of specific DNA molecules from extreme low concentration in sample solution. LDH immobilized through solvent swelling and plasma treatment on the polymer surface captured

Chia-Hao Chan; Jem-Kun Chen; Feng-Chih Chang

2008-01-01

154

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

PubMed Central

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

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

1995-01-01

155

A comparative study of two methods for the isolation of human leucocytes for DNA extraction.  

PubMed

The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis. PMID:2090888

Lim, L H; Ton, S H; Cheong, S K

1990-06-01

156

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

SciTech Connect

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

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

1991-07-01

157

[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 ISO 15189 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 ISO 15189 and the accreditation technical guide in Human Health SH-GTA-04. PMID:24113450

Harlé, Alexandre; Lion, Maëva; Husson, Marie; Dubois, Cindy; Merlin, Jean-Louis

2013-01-01

158

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 14.22±0.135mg/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

159

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

Microsoft Academic Search

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

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

2007-01-01

160

Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures  

Microsoft Academic Search

The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol\\/chloroform>NaI-single tube method>proteinase K digestion equal to amplification of native sera and

Klaus Hamprecht; Elfriede Mikeler; Gerhard Jahn

1997-01-01

161

Evaluation of four DNA extraction methods for the detection of Echinococcus granulosus genotype 1  

PubMed Central

Aim The aim of this survey was to compare four DNA extraction methods from Iranian sheep strain E.granulosus isolates. Background Cystic echinococcosis (CE) caused by the metacestode of the dog tapeworm Echinococcus spp., is a global zoonotic infection which is economically important and constitutes a major threat to public health in many countries. Strains characterization is essential for the establishment of a preventive and control strategy in every endemic area. Patients and methods Forty five infected organs from cattle, sheep and goat were collected from different abattoirs of Iran. All cysts were examined by microscopic observation of protoscoleces. For each cyst, protoscoleces were aspirated and DNA of each cyst was extracted with 4 different methods including tissue Kit extraction, modified Cinnagen extraction kit, Phenol-chloroform (Sambrook1999) and modified Phenol chloroform methods. Efficiency of the DNA was determined by degree of success in PCR amplification. Results Cinnagen modified extraction and modified Phenol chloroform methods were equally effective and superior to other methods after DNA electrophoresis and PCR reaction. Inhibition was observed in PCR with DNA isolated from protoscoleces, and a 1/100 dilution was able to alleviate this problem with DNA extracted. Conclusion The result of this study show that the quality of extracted DNA using modified Cinnagen extraction kit and modified phenol–chloroform are very high and gave identical results after RCR reaction using 12S rRNA gene. Further evaluation is required for its utilization in other clinical specimens.

Roshani, Mohammad; Lahmi, Farhad; Mojarad, Ehsan Nazemalhosseini

2011-01-01

162

A high volume extraction and purification method for recovering DNA from human bone.  

PubMed

DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow(®) (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex(®) ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8ng±1 to 900ng±159 of DNA compared with the organic method ranging from 0.5ng±0.9 to 855ng±156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time. PMID:24997320

Marshall, Pamela L; Stoljarova, Monika; Schmedes, Sarah E; King, Jonathan L; Budowle, Bruce

2014-09-01

163

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.

2014-01-01

164

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

PubMed

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

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

2012-08-15

165

Flexibility of the B-DNA backbone: effects of local and neighbouring sequences on pyrimidine-purine steps.  

PubMed Central

The structurally correlated dihedral angles epsilon and zeta are known for their large variability within the B-DNA backbone. We have used molecular modelling to study both energetic and mechanical features of these variations which can produce BI/BII transitions. Calculations were carried out on DNA oligomers containing either YpR or RpY dinucleotides steps within various sequence environments. The results indicate that CpA and CpG steps favour the BI/BII transition more than TpA or any RpY step. The stacking energy and its intra- and inter-strand components explain these effects. Analysis of neighbouring base pairs reveals that BI/BII transitions of CpG and CpA are easiest within (Y)n(R)n sequences. These can also induce a large vibrational amplitude for TpA steps within the BI conformation.

Bertrand, H; Ha-Duong, T; Fermandjian, S; Hartmann, B

1998-01-01

166

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

PubMed

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

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

2005-12-28

167

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

PubMed Central

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

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

2008-01-01

168

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

PubMed

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

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

2014-10-01

169

EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES  

PubMed Central

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

de Oliveira, Caio Fernando; Paim, Thiago Galvao da Silva; Reiter, Keli Cristine; Rieger, Alexandre; D'azevedo, Pedro Alves

2014-01-01

170

Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples.  

PubMed

Francisella tularensis is the etiologic agent of the zoonotic disease tularemia and is thought to be maintained in the environment principally by various terrestrial and aquatic vertebrate animals. The organism is known to persist in water or mud for long periods of time and Francisella-specific DNA has been identified from water and soil. To gain a better understanding of the ecology and epidemiology of F. tularensis, it will be important to further explore its distribution in the environment. Therefore, methods must be established to efficiently extract Francisella-specific DNA from the soil and be able to eliminate potential PCR inhibitors. Thus, we evaluated five commercial DNA extraction kits for their ability to recover F. tularensis-specific DNA from soil samples and eliminate potential PCR inhibitors. The kits evaluated included the Puregene DNA purification kit, QIAamp Stool Mini kit, Epicentre Biotech SoilMaster DNA extraction kit, and the UltraClean and PowerMax soil DNA isolation kits from MoBio. Soil samples were spiked with gamma-irradiated F. tularensis SHU-4 strain (corresponding to a range from 10 to 10(5)CFU). Spiked samples were extracted with each kit and evaluated using a F. tularensis-specific real-time PCR assay and an internal positive control assay that measures the presence of potential PCR inhibitors. DNA extraction using the UltraClean and PowerMax kits resulted in the most consistently positive results at the lowest limit of detection (20 and 100CFU/g soil, respectively) for all soil types tested, suggesting that these kits can provide the most sensitive methods for extracting F. tularensis from environmental soil samples. Processing time and cost were also evaluated. PMID:17011748

Whitehouse, Chris A; Hottel, Hannah E

2007-04-01

171

A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding  

Microsoft Academic Search

Genomic DNA extraction protocols generally require the use of expensive and hazardous reagents necessary for decontamination\\u000a of phenolic compounds from the extracts. In addition, they are lengthy, hindering large-scale sample extractions necessary\\u000a for high-throughput analyses. Here we describe a simple, time and cost-efficient method for genomic DNA extraction from insects.\\u000a The extracted DNA was successfully used in a Polymerase Chain

Venu M. Margam; Emma W. Gachomo; John H. Shukle; Oluwole O. Ariyo; Manfredo J. Seufferheld; Simeon O. Kotchoni

2010-01-01

172

Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs  

Microsoft Academic Search

The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of

Edna S. Miazato Iwamura; José Arnaldo Soares-Vieira; Marcelo Souza Silva; Karina S. Funabashi; Carla D. Godoy

2009-01-01

173

Extraction of DNA from Formalin-Fixed Cetacean Tissues.  

National Technical Information Service (NTIS)

Procedures to obtain DNA from formalin-preserved samples exist; most have been based on paraffin-embedded tissues prepared for histological examination. Few studies have demonstrated the ability to obtain DNA directly from formalin-preserved tissues locat...

C. A. LeDuc K. M. Robertson P. A. Morin R. G. LeDuc

2007-01-01

174

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

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

175

Validation of a viral and bacterial inactivation step during the extraction and purification process of porcine collagen.  

PubMed

In the last few years, regulations for biomolecule production, and especially for extraction and purification of animal molecules such as collagen, have been reinforced to ensure the sanitary safety of the materials. To be authorized to market biomaterials based on collagen, manufacturers now have to prove that at least one step of their process is described in guidelines to inactivate prion, viruses, and bacteria. The present study focuses on the inactivation step performed during the extraction and purification of porcine type I atelocollagen. We chose to determine the reduction factor of a 1 M NaOH step on porcine parvovirus and four bacterial strains inactivation. During the extraction step, we deliberately inoculated the collagen suspension with the different microorganisms tested. Then, 1 M NaOH was added to the suspension for 1 hour at 20 degrees C. We demonstrated that this treatment totally inactivated S. aureus, P. aeruginosa, C. albicans and A. niger which are bacterial strains responsible of severe human pathology. The reduction factors reached more than 4 logs for B. cereus spores and 4 logs for the porcine parvovirus. are encouraging as those two microorganisms are known to be very resistant to inactivation. PMID:17611295

Forest, P; Morfin, F; Bergeron, E; Dore, J; Bensa, S; Wittmann, C; Picot, S; Renaud, F N R; Freney, J; Gagnieu, C

2007-01-01

176

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

PubMed Central

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

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

2013-01-01

177

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

178

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

PubMed

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

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

2014-02-01

179

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

180

DNA extraction protocol for biological ingredient analysis of liuwei dihuang wan.  

PubMed

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

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

2014-06-01

181

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

182

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

PubMed

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 H(2)O(2)-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 H(2)O(2) for 1 hour. DNA damage was significantly induced by H(2)O(2) 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

Onay-Uçar, Evren; Erol, Ozlem; Kandemir, Ba?ak; Merto?lu, Elif; Karagöz, Ali; Arda, Nazl?

2012-01-01

183

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.

Onay-Ucar, Evren; Erol, Ozlem; Kandemir, Basak; Mertoglu, Elif; Karagoz, Ali; Arda, Nazl?

2012-01-01

184

Aptamer Selection Express: A Rapid Single-Step Selection of Double Stranded DNA Capture Elements (Briefing Charts). Interim Report.  

National Technical Information Service (NTIS)

Advantages of aptamers. ALISA, Where we came from. Dot Blot Format, Other possibilities. One step Quantum Dot De-quenching Assay Why we need a double-stranded DNA aptamer. Comparing SELEX to Aptamer Selection Express (ASExpP). Reagentless electronic senso...

E. Holwitt J. Kiel M. Fan V. Sorola

2009-01-01

185

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

NASA Astrophysics Data System (ADS)

Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H 2O 2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

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

2011-02-01

186

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

PubMed

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

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

2011-01-01

187

One-step polymer surface modification for minimizing drug, protein, and DNA adsorption in microanalytical systems.  

PubMed

The non-specific adsorption of dissolved analytes strongly reduces the sensitivity and reliability in polymer microanalytical systems. Here, a one-step aqueous phase procedure modifies polymer material surfaces to strongly reduce their non-specific adsorption of a broad range of organic analytes including hydrophobic and hydrophilic drugs (0.23 < ClogP < 8.95), small and large proteins (insulin, albumin, IgG), and DNA. The coating is shown to limit the adsorption of even highly hydrophobic drugs (ClogP > 8) in their pharmaceutically relevant concentration range ?100 nM. The low adsorption is mediated by photochemical conjugation, where polyethylene glycol (PEG) polymers in aqueous solution are covalently bound to the surface by UV illumination of dissolved benzophenone and a functionalized PEG. The method can coat the interior of polymer systems made from a range of materials commonly used in microanalytical systems, including polystyrene (PS), cyclic olefin copolymer (COC), liquid crystalline polymer (LCP), and polyimide (PI). PMID:23254780

Larsen, Esben Kjær Unmack; Larsen, Niels B

2013-02-21

188

Implementation and validation of the Teleshake unit for DNA IQ robotic extraction and development of a large volume DNA IQ method.  

PubMed

Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ methods previously developed for the Biomek 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science. PMID:20345792

Grubb, Jennifer C; Horsman-Hall, Katie M; Sykes, Karen L V; Schlisserman, Rebecca A; Covert, Vanessa M; Rhee, Han Na; Ban, Jeffrey D; Greenspoon, Susan A

2010-05-01

189

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

Microsoft Academic Search

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

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

2009-01-01

190

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

Microsoft Academic Search

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

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

2007-01-01

191

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

Microsoft Academic Search

Sarcoptes mite from collection to DNA extraction forms the cornerstone for studies on Sarcoptes scabiei. Whilst the new science era took a shy leap into the different facets of mite studies, the cornerstone was almost entirely\\u000a neglected. Mite collection, cleaning, storage and DNA extraction were, basically, humble attempts to extrapolate, adapt, modify\\u000a or ‘pirate’ those existing methods to the peculiarities

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

2009-01-01

192

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

PubMed Central

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

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

2013-01-01

193

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 (4-6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability. PMID:24294115

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

2013-12-01

194

Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues.  

PubMed

The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50 years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations ? 10.0 ng/?l were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72 h instead of 1-2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280 nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR). PMID:23603300

Paireder, Stefan; Werner, Bettina; Bailer, Josef; Werther, Wolfgang; Schmid, Erich; Patzak, Beatrix; Cichna-Markl, Margit

2013-08-15

195

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

Microsoft Academic Search

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

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

196

Evaluation of three DNA extraction protocols for forensic STR typing after laser capture microdissection.  

PubMed

In forensic sciences, short tandem repeat (STR) analysis is a valuable tool in identifying the donor(s) of biological stains. Laser capture microdissection (LCM) can be used as a cell separating technique to isolate specific cell types in mixed samples. An important challenge lies in the development of a DNA isolation method appropriate for laser microdissected cells, as these samples usually contain minute amounts of cells. In this study three different DNA isolation methods for LCM collected cells were compared. The PicoPure DNA extraction method outperformed both other methods (IQ™ system and short alkaline method). Consequently, the minimal number of LCM collected cells necessary for STR typing was determined. Using the PicoPure DNA extraction method, full DNA profiles could be obtained from as little as 10 cells. Nevertheless, despite the occurrence of allelic drop out in some of the samples, lower amounts of cells gave rise to useful DNA profiles. PMID:21727054

Vandewoestyne, Mado; Van Nieuwerburgh, Filip; Van Hoofstat, David; Deforce, Dieter

2012-03-01

197

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

PubMed

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

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

2013-01-01

198

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

PubMed

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 SK(bi) ? 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 SK(uni) < 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. PMID:24089739

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

2013-09-28

199

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

PubMed

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

Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

2012-01-01

200

Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva  

Microsoft Academic Search

Background: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). Methods: Saliva was collected from

Daniel P. K Ng; David Koh; Serena G. L Choo; Vivian Ng; Qiuyun Fu

2004-01-01

201

STR typing of ancient DNA extracted from hair shafts of Siberian mummies  

Microsoft Academic Search

The aim of this study was to determine if ancient hair shafts could be suitable for nuclear DNA analysis and to develop an efficient and straightforward protocol for DNA extraction and STR typing of ancient specimens. The developed method was validated on modern and forensic samples and then successfully applied on ancient hairs collected from Siberian mummies dating from the

S. Amory; C. Keyser; E. Crubézy; B. Ludes

2007-01-01

202

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

Microsoft Academic Search

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

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

2006-01-01

203

Improved method facilitates reliable APOE genotyping of genomic DNA extracted from formaldehyde-fixed pathology specimens  

Microsoft Academic Search

Apolipoprotein E (APOE) genotyping of genomic DNA extracted from formaldehyde-fixed specimens is cumbersome: there is not only a low yield or failure of PCR amplification (presumably due to degradation of DNA in the formaldehyde-fixed and paraffin-embedded tissue), but the standard method also involves the separation of DNA fragments as small as 48, 72, 81 and 91 bp requiring high-yield PCR

Estifanos Ghebremedhin; Heiko Braak; Eva Braak; Jürgen Sahm

1998-01-01

204

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

PubMed Central

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

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

2012-01-01

205

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil  

PubMed Central

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

Lin, Jianghai; Kennedy, Stephen H.; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T.

2009-01-01

206

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil.  

PubMed

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp. PMID:19698695

Lin, Jianghai; Kennedy, Stephen H; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W; Xu, Anlong; Zondervan, Krina T

2009-12-15

207

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.

Pandey, Manjula; Patel, Smita S.

2014-01-01

208

Automated serial extraction of DNA and RNA from biobanked tissue specimens  

PubMed Central

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

2013-01-01

209

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

210

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

PubMed Central

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

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

2012-01-01

211

A DNA-extraction and polymerase-chain-reaction microchip using magnetic beads and thermo-pneumatic valves  

Microsoft Academic Search

In this work, we develop a miniaturized system for DNA extraction and polymerase chain reaction (PCR). DNA is extracted from whole blood sample by using silica coated magnetic beads. Electromagnetic actuation is employed to manipulate the magnetic beads for transporting DNA molecules in fluidic channels. Thermo-pneumatic valves with low temperature elevation are integrated into the microfluidic chip for isolating\\/releasing fluidic

Bonnie T. Chia; Xing-Ying Yang; Ming-Yuan Cheng; Yao-Joe Yang

2010-01-01

212

Utility of arsenic-treated bird skins for DNA extraction  

Microsoft Academic Search

Background  Natural history museums receive a rapidly growing number of requests for tissue samples from preserved specimens for DNA-based\\u000a studies. Traditionally, dried vertebrate specimens were treated with arsenic because of its toxicity and insect-repellent\\u000a effect. Arsenic has negative effects on in vivo DNA repair enzymes and consequently may inhibit PCR performance. In bird collections, foot pad samples are often requested\\u000a since

Till Töpfer; Anita Gamauf; Elisabeth Haring

2011-01-01

213

Utility of arsenic-treated bird skins for DNA extraction  

PubMed Central

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

2011-01-01

214

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 kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.

Baker, Erin J.; Kellogg, Christina A.

2014-01-01

215

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

PubMed

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

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

2013-01-01

216

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.

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

2014-01-01

217

Cell-free fetal DNA plasma extraction and real-time polymerase chain reaction quantification.  

PubMed

Isolation, quantification, and genetic analysis of circulating plasma DNA have clinical applications in prenatal diagnosis, oncology, organ transplantation, posttrauma monitoring, and infectious disease. Recent technology has allowed the rapid isolation and purification of DNA from whole blood, plasma, serum, buffy coat, tissues, stool, and urine. With the advent of real-time polymerase chain reaction (PCR) amplification, extracted DNA not only can be easily identified to aid in clinical diagnoses, but also can be readily quantified to analyze ongoing clinical dynamics and aid in the medical prognoses of patients. Historically, identification of unique cell-free fetal DNA sequences has relied on the detection of paternally specific Y chromosome sequences owing to their relative ease in identification. However, any DNA sequence that is unique to the fetus has the potential to be amplified and quantified using real-time PCR. Our laboratory specializes in extraction of fetal DNA from maternal plasma with subsequent quantification with real-time PCR of paternally inherited sequences, such as the Y chromosome gene, SRY. The successful isolation and quantification of this DNA from plasma is dependent on three distinct protocols: plasma harvesting from whole blood, DNA extraction from cell-free plasma, and real-time PCR amplification and quantification of the SRY sequence. PMID:17876076

Maron, Jill L; Johnson, Kirby L; Bianchi, Diana W

2007-01-01

218

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

PubMed Central

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

2010-01-01

219

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

Microsoft Academic Search

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

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

2003-01-01

220

Single Molecule FRET Analysis of the 11 Discrete Steps of a DNA Actuator.  

PubMed

DNA hybridization allows the design and assembly of dynamic DNA-based molecular devices. Such structures usually accomplish their function by the addition of fuel strands that drive the structure from one conformation to a new one or by internal changes in DNA hybridization. We report here on the performance and robustness of one of these devices by the detailed study of a dynamic DNA actuator. The DNA actuator was chosen as a model system, as it is the device with most discrete states to date. It is able to reversibly slide between 11 different states and can in principle function both autonomously and nonautonomously. The 11 states of the actuator were investigated by single molecule Förster Resonance Energy Transfer (smFRET) microscopy to obtain information on the static and dynamic heterogeneities of the device. Our results show that the DNA actuator can be effectively locked in several conformations with the help of well-designed DNA lock strands. However, the device also shows pronounced static and dynamic heterogeneities both in the unlocked and locked modes, and we suggest possible structural models. Our study allows for the direct visualization of the conformational diversity and movement of the dynamic DNA-based device and shows that complex DNA-based devices are inherently heterogeneous. Our results also demonstrate that single molecule techniques are a powerful tool for structural dynamics studies and provide a stringent test for the performance of molecular devices made out of DNA. PMID:24857342

Hildebrandt, Lasse L; Preus, Søren; Zhang, Zhao; Voigt, Niels V; Gothelf, Kurt V; Birkedal, Victoria

2014-06-25

221

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

EPA Science Inventory

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

222

D Object Coordinates Extraction by Radargrammetry and Multi Step Image Matching  

NASA Astrophysics Data System (ADS)

Nowadays by high resolution SAR imaging systems as Radarsat-2, TerraSAR-X and COSMO-skyMed, three-dimensional terrain data extraction using SAR images is growing. InSAR and Radargrammetry are two most common approaches for removing 3D object coordinate from SAR images. Research has shown that extraction of terrain elevation data using satellite repeat pass interferometry SAR technique due to atmospheric factors and the lack of coherence between the images in areas with dense vegetation cover is a problematic. So the use of Radargrammetry technique can be effective. Generally height derived method by Radargrammetry consists of two stages: Images matching and space intersection. In this paper we propose a multi-stage algorithm founded on the combination of feature based and area based image matching. Then the RPCs that calculate for each images use for extracting 3D coordinate in matched points. At the end, the coordinates calculating that compare with coordinates extracted from 1 meters DEM. The results show root mean square errors for 360 points are 3.09 meters. We use a pair of spotlight TerraSAR-X images from JAM (IRAN) in this article.

Eftekhari, A.; Ghannadi, M. A.; Motagh, M.; Saadat Seresht, M.

2013-09-01

223

A Novel Two-Step-Deformation Active Contour with Application to Facial Features Extraction  

Microsoft Academic Search

In allusion to the boundary leaking problem of traditional active contours when used to extract facial features, two kinds of local information are introduced to enhance the weak boundary, then to build new external force for active contours. The prior knowledge of the local shape of the facial features is incorporated into the evolution of active contour. The snake contour

Hua Zhang; Yuanquan Wang

2009-01-01

224

Microbes on building materials--evaluation of DNA extraction protocols as common basis for molecular analysis.  

PubMed

The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. PMID:23063637

Ettenauer, Jörg D; Piñar, Guadalupe; Lopandic, Ksenija; Spangl, Bernhard; Ellersdorfer, Günther; Voitl, Christian; Sterflinger, Katja

2012-11-15

225

Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting.  

PubMed

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T(m)). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T(m), as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification. PMID:21119928

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

2010-12-01

226

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts  

PubMed Central

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) ?, ? or ?. Base excision repair was also proficient without Pol?. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Pol? (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Pol? (pol3-1) or Pol? (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Pol? and Pol?, respectively. Deleting the polymerase catalytic domain of Pol? (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol?, but not by purified yeast Pol?. These results suggest that efficient repair synthesis of yeast NER requires both Pol? and Pol? in vitro, and that the low fidelity Pol? is not accessible to repair synthesis during NER.

Wu, Xiaohua; Guo, Dongyu; Yuan, Fenghua; Wang, Zhigang

2001-01-01

227

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

228

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

229

Protective effect of enzymatic extracts from microalgae against DNA damage induced by H2O2.  

PubMed

The enzymatic extracts from seven species of microalgae (Pediastrum duplex, Dactylococcopsis fascicularis, Halochlorococcum porphyrae, Oltmannsiellopsis unicellularis, Achnanthes longipes, Navicula sp. and Amphora coffeaeformis) collected from three habitats (freshwater, tidal pool, and coastal benthic) at Jeju Island in Korea were investigated for their antioxidant activity. Of the extracts tested, the AMG 300 L (an exo 1, 4-alpha-D-glucosidase) extract of P. duplex, the Viscozyme extract of Navicula sp., and the Celluclast extract of A. longipes provided the most potential as antioxidants. Meanwhile, the Termamyl extract of P. duplex in an H(2)O(2) scavenging assay exhibited an approximate 60% scavenging effect. In this study, we report that the DNA damage inhibitory effects of P. duplex (Termamyl extract) and D. fascicularis (Kojizyme extract) were nearly 80% and 69% respectively at a concentration of 100 microg/ml. Thus, it is suggested that the microalgae tested in this study yield promising DNA damage inhibitory properties on mouse lymphoma L 5178 cells that are treated with H(2)O(2). Therefore, microalgae such as P. duplex may be an excellent source of naturally occurring antioxidant compounds with potent DNA damage inhibition potential. PMID:17520314

Karawita, Rohan; Senevirathne, Mahinda; Athukorala, Yasantha; Affan, Abu; Lee, Young-Jae; Kim, Se-Kwon; Lee, Joon-Baek; Jeon, You-Jin

2007-01-01

230

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 São 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

231

DNA Extraction from Nocardia Species for Special Genes Analysis Using PCR  

PubMed Central

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

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

2014-01-01

232

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

NASA Astrophysics Data System (ADS)

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

Wang, Haijing; Kinzelbach, Wolfgang

2010-05-01

233

A simple method to determine bioethanol content in gasoline using two-step extraction and liquid scintillation counting.  

PubMed

A simple method for determining bioethanol content in gasoline containing bioethanol (denoted as E-gasoline in this study) is urgently required. Liquid scintillation counting (LSC) was employed based on the principle that (14)C exists in bioethanol but not in synthetic ethanol. Bioethanol was extracted in two steps by water from E-gasoline containing 3% (E3) or 10% (E10) bioethanol. The (14)C radioactivity was measured by LSC and converted to the amount of bioethanol. The bioethanol content in E-gasoline was determined precisely from the partition coefficient in the extraction and the amount of bioethanol in the water phases: 2.98+/-0.10% for E3 and 10.0+/-0.1% for E10 (means+/-SD; n=3). It appears that this method can be used to determine bioethanol content in E-gasoline quickly and easily. PMID:19577920

Yunoki, Shunji; Saito, Masaaki

2009-12-01

234

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

235

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

PubMed

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

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

2011-02-01

236

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.  

PubMed

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated. PMID:24766813

Liu, Shixin; Chistol, Gheorghe; Hetherington, Craig L; Tafoya, Sara; Aathavan, K; Schnitzbauer, Joerg; Grimes, Shelley; Jardine, Paul J; Bustamante, Carlos

2014-04-24

237

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

PubMed

Currently, detection of DNA hybridization using fluorescence-based detection technique requires expensive optical systems and complex bioinformatics tools. Hence, the development of new low cost devices that enable direct and highly sensitive detection stimulates a lot of research efforts. Particularly, devices based on silicon nanowires are emerging as ultrasensitive electrical sensors for the direct detection of biological species thanks to their high surface to volume ratio. In this study, we propose innovative devices using step-gate polycrystalline silicon nanowire FET (poly-Si NW FETs), achieved with simple and low cost fabrication process, and used as ultrasensitive electronic sensor for DNA hybridization. The poly-SiNWs are synthesized using the sidewall spacer formation technique. The detailed fabrication procedure for a step-gate NWFET sensor is described in this paper. No-complementary and complementary DNA sequences were clearly discriminated and detection limit to 1 fM range is observed. This first result using this nano-device is promising for the development of low cost and ultrasensitive polysilicon nanowires based DNA sensors compatible with the CMOS technology. PMID:22841443

Wenga, G; Jacques, E; Salaün, A-C; Rogel, R; Pichon, L; Geneste, F

2013-02-15

238

Black tea extract: a supplementary antioxidant in radiation-induced damage to DNA and normal lymphocytes.  

PubMed

Myriad research has contributed significantly toward the understanding and identification of health benefits stemming from tea polyphenols and many other naturally occurring flavonoids present in fruits and vegetables. These flavonoids are known to mitigate reactive oxygen species-induced damage by scavenging them. In this study, hot-water black tea extract rich in flavonoids is evaluated as a supplementary antioxidant. The antioxidant efficacy of black tea extract was investigated by evaluating radioprotection conferred to pBR322 DNA, calf thymus DNA, and normal lymphocytes during gamma irradiation. The protection was measured by gel electrophoresis, fluorimetric study, cell viability assay, cytokinesis-blocked micronuclei assay, and comet assay. The 2,2-diphenyl-1-picrylhydrazyl scavenging ability of the tea extract used increased in a dose-dependent manner (IC50: 182.45 µg/mL). Positive correlation of radioprotection with antioxidant activity of black tea extract was observed in all systems. Maximum protection against radiation-induced damage was observed in pBR322 DNA and calf thymus DNA at ?200 µg/mL of black tea extract. At a dose of black tea extract as low as 5 µg/mL, efficient radioprotection was observed in normal lymphocytes, which is encouraging and can be tested in the future as a natural antioxidant supplement during radiotherapy. PMID:23216640

Ghosh, Debjani; Pal, Sandip; Saha, Chabita; Chakrabarti, Amit Kumar; Datta, Salil C; Dey, Subrata Kumar

2012-01-01

239

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

PubMed Central

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

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

2012-01-01

240

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

NASA Astrophysics Data System (ADS)

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

Sharma, Ajeet K.; Chowdhury, Debashish

2013-09-01

241

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

NASA Astrophysics Data System (ADS)

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

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

2011-05-01

242

A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples  

Microsoft Academic Search

Isolation of amplifiable genomic DNA is a prerequisite for the genetic assessment of diseases and disease susceptibility in farm animals. Milk somatic cells are a practical, animal friendly and cost-effective source of genomic DNA in milking ruminants. In this study, six different DNA extraction methods were optimized, evaluated and compared for the isolation of DNA from ovine milk samples. Methods

Androniki Psifidi; Chrysostomos I. Dovas; Georgios Banos

2010-01-01

243

DNA Extraction by Isotachophoresis in a Microfluidic Channel  

Microsoft Academic Search

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

2011-01-01

244

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

245

Extraction of viral DNA from erythrocytes of swine with acute African swine fever.  

PubMed

The preparation of wild-type African swine fever (ASF) virus DNA from small amounts of viremic blood from acutely febrile pigs is outlined. The extracted DNA is viral and not host-cell DNA, because of specific homology with cell culture grown and purified ASF virus and because no DNA bands are obtained with an equal amount of nonviremic pig blood. Thus, in the absence of suitable serologic methods for strain identification, it is now possible to catalogue wild-type isolates by characteristic DNA restriction patterns. The wild-type virus genome contains terminal single-stranded DNA cross-links and has the largest genome size (180 kilobase pairs) reported for the ASF virus. Experimental passage of the virus in contact-infected pigs and buffy coat cultures appears to confirm the stable nature of the ASF genome in the field. PMID:6331232

Wesley, R D; Quintero, J C; Mebus, C A

1984-06-01

246

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

PubMed Central

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

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

2013-01-01

247

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

PubMed Central

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

2012-01-01

248

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

Microsoft Academic Search

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

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

2010-01-01

249

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

PubMed

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

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

2006-12-01

250

A robust universal method for extraction of genomic DNA from bacterial species.  

PubMed

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCI. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method f r large-scale DNA isolation fromvarious bacterial species. PMID:21058509

Atashpaz, Sina; Khani, Sajjad; Barzegari, Abolfazl; Barar, Jaleh; Vahed, Sepideh Zununi; Azarbaijani, Reza; Omidi, Yadollah

2010-01-01

251

Single-step extraction and cleanup of bisphenol A in soft drinks by hemimicellar magnetic solid phase extraction prior to liquid chromatography/tandem mass spectrometry.  

PubMed

Hemimicelles of tetradecanoate chemisorbed onto magnetic nanoparticles (MNPs) are here proposed as a sorbent for the single-step extraction and cleanup of bisphenol A (BPA) in soft drinks. The purpose of this work was to develop a simple, rapid and low-cost sample treatment suitable to assess the human exposure to BPA from this type of high consumption food. The nanoparticles were easily coated by mixing commercially available magnetite of 20-30 nm mean particle diameter with tetradecanoate at 85°C for 30 min. The extraction/cleanup procedure involved stirring the samples (3 mL) with 200mg of tetradecanoate-coated MNPs for 20 min, isolating the sorbent with a Nd-Fe-B magnet and eluting BPA with methanol. The extraction efficiency was not influenced by salt concentrations up to 1M and pH values over the range 4-9. No cleanup of the extracts was needed, and the method proved matrix-independent. The extracts were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Quantitation was performed by internal standard calibration using BPA-(13)C12. The limit of quantitation obtained for the method, 0.03 ng mL(-1), was below the usual range of concentrations reported for BPA in soft drinks (0.1-3.4 ng mL(-1)). The proposed method was successfully applied to the determination of BPA in different samples acquired from various supermarkets in southern Spain; the concentrations found ranged from 0.066 to 1.08 ng mL(-1). Recoveries from samples spiked with 0.33 ng mL(-1) of BPA ranged from 91% to 105% with relative standard deviations from 3% to 8%. PMID:23639396

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

2013-05-17

252

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

253

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

PubMed

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

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

2012-07-29

254

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation  

PubMed Central

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2-7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2-7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2-7 when cells entered S-phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.

van Deursen, Frederick; Sengupta, Sugopa; De Piccoli, Giacomo; Sanchez-Diaz, Alberto; Labib, Karim

2012-01-01

255

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

PubMed Central

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

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

2011-01-01

256

Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA  

Microsoft Academic Search

Carboxyl-coated magnetic nanoparticles (MNPs) were used to demonstrate dual functionality: isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled (sc) form of plasmid DNA (pDNA) from agarose gel. These MNPs were attached with 5?-NH2-tagged oligo-(dT)25 primer and were used to isolate mRNA from breast cancer cells. The isolated mRNA was used for amplification of ?-actin to

Tapasree Roy Sarkar; Joseph Irudayaraj

2008-01-01

257

Release of 7-methylguanine Residues from Alkylated DNA by Extracts of Micrococcus luteus and Escherichia coli  

Microsoft Academic Search

Cell extracts from Micrococcus luteus release both free 3-methyladenine and free 7-methylguanine from alkylated DNA. The glycosylase activity responsible for the liberation of 7-methylguanine is not 3-methyladenine-DNA glycosylase, which, when purified, does not liberate it. Furthermore, the heat inactivation rates of the two enzymatic activities are different. The release of 7-methylguanine by chemical depurination of ethanol-soluble oligonucleotides has been ruled

Jacques Laval; Josiane Pierre; Francoise Laval

1981-01-01

258

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

Microsoft Academic Search

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

Scott O. Rogers; Arnold J. Bendich

1985-01-01

259

The harnessing of peptide–monolith constructs for single step plasmid DNA purification  

Microsoft Academic Search

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as

Ying Han; Guanlin You; Leonard K. Pattenden; Gareth M. Forde

2010-01-01

260

A comparison of methods for total community DNA preservation and extraction from various thermal environments.  

PubMed

The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments. PMID:18633656

Mitchell, Kendra R; Takacs-Vesbach, Cristina D

2008-10-01

261

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

262

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

PubMed

The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2. PMID:15630179

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

2004-01-01

263

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

PubMed Central

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

2011-01-01

264

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against ?-Bungarotoxin  

PubMed Central

Background Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. Principal Findings Herein, we present a simple one-step selection of DNA aptamers against ?-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. Conclusion We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

Lauridsen, Lasse H.; Shamaileh, Hadi A.; Edwards, Stacey L.; Taran, Elena; Veedu, Rakesh N.

2012-01-01

265

Two simple techniques for the safe Sarcoptes collection and individual mite DNA extraction  

Microsoft Academic Search

Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency\\u000a and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic\\u000a skin samples (crusts or deep skin scrapings). Its

Dominga Soglia; Luisa Rambozzi; Sandra Maione; Veronica Spalenza; Stefano Sartore; Samer Alasaad; Paola Sacchi; Luca Rossi

2009-01-01

266

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

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

267

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.

Knipscheer, Puck; Raschle, Markus; Scharer, Orlando D.; Walter, Johannes C.

2014-01-01

268

Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors.  

PubMed Central

The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity. These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase. This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J. Laffan and W. Firshein, J. Bacteriol. 169:2819-2827, 1987). Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation. Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives. Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase. Some regulatory mechanisms of the kinase system may also be retained by the complex. Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex. Images

Laffan, J J; Skolnik, I L; Hadley, D A; Bouyea, M; Firshein, W

1990-01-01

269

Magnetic bead-based solid phase for selective extraction of genomic DNA  

Microsoft Academic Search

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media.

Marie J. Archer; Baochuan Lin; Zheng Wang; David A. Stenger

2006-01-01

270

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

Microsoft Academic Search

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

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

2004-01-01

271

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 65°C±2°C 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 (1.25×10?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.

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

2013-01-01

272

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 65°C±2°C 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 (1.25×10(-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

273

Extraction of helium from individual interplanetary dust particles by step-heating  

NASA Technical Reports Server (NTRS)

Fragments from 20 individual particles, collected in the earth's stratosphere and believed to be interplanetary dust particles (IDPs), were subjected to step-heating to see if differences in the release pattern for He-4 could be observed which might provide clues to the origin of the particles. Comparisons were made to the release pattern for 18 individual lunar surface grains heated in the same manner. Twelve of the IDP fragments contained an appreciable amount of He-4, 50 percent of which was released by the time the particles were heated to approximately 630 C. For the 18 individual lunar grains, the corresponding average temperature was 660 C. The He-3/He-4 ratios found for these fragments agreed well with those found for deep Pacific magnetic fines believed to be of extraterrestrial origin, and were comparable to those which have been observed for the solar wind and lunar surface soil grains. Four of the IDP fragments contained appreciably less He-4, and this was released at a higher temperature. The remaining four fragments had too little He-4 to permit a determination. From Flynn's analyses of the problem of the heating of IDPs in their descent in the atmosphere, the present results suggest that the parent IDPs of the 12 particles which contained an appreciable amount of He-4 suffered very little heating in their descent and are likely of asteroidal origin, although one cannot rule out the possibility that at least some of them had a cometary origin and entered the earth's atmosphere at a grazing angle.

Nier, A. O.; Schlutter, D. J.

1992-01-01

274

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

PubMed Central

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

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

2006-01-01

275

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation  

PubMed Central

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (?1.0 µM), or ionizing radiation (IR) (?15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.

Wang, Xiang; Guan, Jun; Hu, Baocheng; Weiss, Robert S.; Iliakis, George; Wang, Ya

2004-01-01

276

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

Microsoft Academic Search

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

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

2007-01-01

277

Evaluation of DNA Extraction and PCR Methods for Detection of Enterocytozoon bienuesi in Stool Specimens  

Microsoft Academic Search

An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration

Ittisak Subrungruang; Mathirut Mungthin; Porntip Chavalitshewinkoon-Petmitr; Ram Rangsin; Tawee Naaglor; Saovanee Leelayoova

2004-01-01

278

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

Microsoft Academic Search

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

Ibrahim Abdullah Alaraidh

279

Patterning of a nanoporous membrane for multi-sample DNA extraction  

Microsoft Academic Search

A novel method for patterning a nanoporous aluminum oxide membrane was developed using SU-8 to allow for extraction of DNA from multiple samples simultaneously. To facilitate the patterning process, the spin curve for SU-8 2015 on the membrane was determined. The correlation profile of the SU-8 spin curve was similar in form to curves of SU-8 spun on silicon wafers,

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

2006-01-01

280

Fidelity of DNA Replication by Extracts of Normal and Malignantly Transformed Human Cells1  

Microsoft Academic Search

To test the hypothesis that a imitator phenotype may be associated with carcinogenesis (L. A. Loeb, Cancer Res., 5\\/: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the effi ciency of mismatch repair in extracts from normal diploid and malig nantly transformed human cells. Included was a diploid fibroblast strain and its transformed derivative, as well

Jayne C. Boyer; David C. Thomas; Veronica M. MÃ; Justin J. McCormick; Thomas A. Kunkel

1993-01-01

281

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)  

Microsoft Academic Search

The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues,

A. Barbaro; P. Cormaci; A. Agostino

2009-01-01

282

Aptamer Selection Express: A Rapid Single-Step Selection of Double- stranded DNA Capture Elements.  

National Technical Information Service (NTIS)

SELEX: Advantages of Aptamers * Aptamers are smaller than antibodies -- ranging from 30 to 50 nucleotides * Do not require either animals or tissue culture for production * Can be synthesized chemically or by PCR * Due to the nature of DNA, they are stabl...

E. A. Holwitt J. L. Kiel M. Fan V. K. Sorola

2009-01-01

283

Fluoroplast-polyaniline-coated adsorbent for one-step isolation of DNA for PCR detection of viral hepatitides (HBV and TTV).  

PubMed

Aims: To demonstrate the effectiveness of application of the adsorbent successively modified with nano-layers of fluoroplast and polyaniline for one-step isolation of DNA of hepatitis B virus and transfusion-transmitted virus from human serum. Materials & Methods: The technique is based on the application of the spin-cartridges containing porous adsorbent for one-step viral DNA isolation from serum followed by polymerase chain reaction. Results: The developed adsorbent was shown to be effective for one-step isolation of viral DNA from serum samples for polymerase chain reaction diagnostics. Conclusion: The effectiveness of the developed adsorbent application for isolation of viral DNA from serum for polymerase chain reaction diagnostics was confirmed in comparison with standard methods. Thus, the facile sample preparation method of viral DNA isolation was elaborated. PMID:24806904

Kapustin, Dmitry V; Prostyakova, Anna I; Zubov, Vitaly P

2014-04-01

284

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

PubMed

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method. PMID:21818364

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

2011-01-01

285

Extraction of RAPD-friendly DNA from Laminaria japonica (Phaeophyta) after enzymatic dissociation of the frozen sporophyte tissues  

Microsoft Academic Search

A new extraction protocol has been developed to obtain high quality DNAfrom Laminaria japonica, which involves enzymatic dissociation ofsporophyte tissues and subsequent elimination of the remainingpolysaccharides with cetyltrimethyl ammonium bromide. Unicells isolatedfrom frozen kelp tissues with alginate lyase prepared from the abalone Haliotis diversicolor were used to extract total DNA; the yield wasapproximately 13 to 22.5 µg DNA g-1 (wet

Y.-J. Hu; Z.-G. Zhou

2001-01-01

286

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

287

Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up.  

PubMed

The measurement of oxidative damage to cellular DNA is a challenging analytical problem requiring highly sensitive and specific methods. In addition, artefactual DNA oxidation during its extraction and subsequent work-up may give rise to overestimated levels of oxidized DNA bases. In the present study, we have used (18)O-labelled 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) as an internal standard to evaluate the extent of artefactual DNA oxidation during the critical steps preceding the measurement. The labelled oxidized purine nucleoside was specifically generated in cellular DNA using the recently available generator of (18)O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the (18)O-oxidized nucleoside. Therefore, the ratio between the two compounds, as measured by high performance liquid chromatography coupled to tandem mass spectrometry, allows an unambiguous comparison of different methodologies. The comparison of different DNA extraction protocols led to the conclusion that artefactual DNA oxidation during the extraction step could be minimized if: (i) nuclei are isolated after cell lysis; (ii) desferrioxamine, a transition metal chelator is added to the different extraction buffers; and (iii) sodium iodide (or alternatively guanidine thiocyanate) is used for DNA precipitation. It was also demonstrated that sodium iodide does not decompose the targeted oxidized purine nucleoside. In addition, three different DNA digestion protocols were evaluated and they were found to give rise to similar results. Using the best-studied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be approximately 0.5 lesions/10(6) DNA nucleosides. PMID:12419840

Ravanat, Jean-Luc; Douki, Thierry; Duez, Pierre; Gremaud, Eric; Herbert, Karl; Hofer, Tim; Lasserre, Lydie; Saint-Pierre, Christine; Favier, Alain; Cadet, Jean

2002-11-01

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 70°C. Under the optimum conditions, the calibration curves showed good linearity (R(2)>0.9994) and precision (relative standard deviation, RSD?2.4%) within the tested ranges. The limits of quantification for 1,3-DCP and 3-MCDP, 1.6 and 1.7 ?g kg(-1), respectively, are far below the established limits in the European and American legislations. The accuracy, precision, linearity, and limits of quantification provided make this analytical method suitable for routine control. The method was applied to the analysis of several toasted bread, snacks, cookies and cereal samples, none of which contained chloropropanols at concentrations above the legislation levels. PMID:21875707

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

2011-09-28

289

Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device.  

PubMed

The evaluation of a micro fluidic system with an integrated silica monolith for performing DNA extraction from limited biological samples has been carried out. Low DNA target concentrations usually require the addition of carrier RNA to ensure desired extraction efficiencies. Here, we demonstrate a micro fluidic extraction system with increasingly efficient extraction performances for biological samples containing <15 ng of total DNA without the need of adding carrier nucleic acids. All extracted DNA showed successful amplification via the polymerase chain reaction demonstrating both the effectiveness of the proposed system at removing potential inhibitors and yielding good quality DNA. The work presented here beneficially identifies reduced sample volumes/concentrations as suitable for processing with respect to downstream analysis by enabling pre-concentration of the biological sample, particularly important when dealing with clinical or forensic specimens. PMID:23062434

Kashkary, Loay; Kemp, Cordula; Shaw, Kirsty J; Greenway, Gillian M; Haswell, Stephen J

2012-10-31

290

Comparison of alkaline lysis with electroextraction and optimization of electric pulses to extract plasmid DNA from Escherichia coli.  

PubMed

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

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

2013-11-01

291

Hippophae leaf extract concentration regulates antioxidant and prooxidant effects on DNA.  

PubMed

Extracts from Hippophae leaves constitute some commonly consumed beverages such as tea and wine. We had developed an extract of Hippophae leaves (SBL-1), which was rich in quercetin, had antimutagenic effects, radioprotective effects, and countered radiation-induced gene conversion in Saccharomyces cerevisiae. This study was designed to investigate the action of SBL-1 on guanine cytosine (GC)-rich nascent and mouse genomic DNA in vitro. The human and mouse liver DNA have about 43% GC content. Our results showed that at small concentration SBL-1 protected nascent as well as genomic DNA, while at large concentration SBL-1 damaged both types of DNA. The concentration of SBL-1 that protected DNA also demonstrated higher free radical scavenging activity. The reducing power of SBL-1 was greater than its free radical scavenging activity. The greater reducing power may have reduced the trace metals present in the SBL-1, leading to generation of hydroxyl radicals via Fenton reaction. The increased proportion of unscavenged hydroxyl radicals with increase in SBL-1 concentration may have been responsible for DNA damage or prooxidant effect of SBL-1 in vitro. This study suggests that the dietary supplements prepared from Hippophae should have low metal content. PMID:22435574

Saini, Manu; Tiwari, Sandhya; Prasad, Jagdish; Singh, Surender; Kumar, M S Yogendra; Bala, Madhu

2010-03-01

292

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

PubMed Central

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

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

2013-01-01

293

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

PubMed

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

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

2013-09-30

294

Effects of growth medium selection on plasmid DNA production and initial processing steps  

Microsoft Academic Search

Cultures of recombinant Escherichia coli containing the plasmid pSV? were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria–Bertani

R. D O’Kennedy; C Baldwin; E Keshavarz-Moore

2000-01-01

295

First steps towards systems radiation biology studies concerned with DNA and chromosome structure within living cells  

Microsoft Academic Search

For the understanding of radiation action on biological systems like cellular macromolecules (e.g., DNA in its higher structures)\\u000a a synergistic approach of experiments and quantitative modelling of working hypotheses is necessary. Further on, the influence\\u000a on calculated results of certain assumptions in such working hypotheses must critically be evaluated. In the present work,\\u000a this issue is highlighted in two aspects

Werner Friedland; Herwig G. Paretzke; Francesca Ballarini; Andrea Ottolenghi; Gregor Kreth; Christoph Cremer

2008-01-01

296

Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip  

Microsoft Academic Search

BACKGROUND: As FTA® cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes ? 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted

Matthew C McClure; Stephanie D McKay; Robert D Schnabel; Jeremy F Taylor

2009-01-01

297

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

Microsoft Academic Search

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

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

2009-01-01

298

One-step DNA-programmed growth of luminescent and biofunctionalized nanocrystals  

NASA Astrophysics Data System (ADS)

Colloidal semiconductor nanocrystals are widely used as lumiphores in biological imaging because their luminescence is both strong and stable, and because they can be biofunctionalized. During synthesis, nanocrystals are typically passivated with hydrophobic organic ligands, so it is then necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water. Finally, biological labels must be added to allow the detection of nucleic acids, proteins and specific cell types. This multistep process is time- and labour-intensive and thus out of reach of many researchers who want to use luminescent nanocrystals as customized lumiphores. Here, we show that a single designer ligand-a chimeric DNA molecule-can controllably program both the growth and the biofunctionalization of the nanocrystals. One part of the DNA sequence controls the nanocrystal passivation and serves as a ligand, while another part controls the biorecognition. The synthetic protocol reported here is straightforward and produces a homogeneous dispersion of nanocrystal lumiphores functionalized with a single biomolecular receptor. The nanocrystals exhibit strong optical emission in the visible region, minimal toxicity and have hydrodynamic diameters of ~6 nm, which makes them suitable for bioimaging. We show that the nanocrystals can specifically bind DNA, proteins or cells that have unique surface recognition markers.

Ma, Nan; Sargent, Edward H.; Kelley, Shana O.

2009-02-01

299

Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination.  

PubMed Central

The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate. Images

Parsons, R L; Prasad, P V; Harshey, R M; Jayaram, M

1988-01-01

300

New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.  

PubMed

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D(3) hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration. PMID:20154109

Sallam, Khalid Ibrahim; Tamura, Noriko; Imoto, Noriko; Tamura, Tomohiro

2010-04-01

301

New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome?  

PubMed Central

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D3 hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.

Sallam, Khalid Ibrahim; Tamura, Noriko; Imoto, Noriko; Tamura, Tomohiro

2010-01-01

302

Mouse sepiapterin reductase: an enzyme involved in the final step of tetrahydrobiopterin biosynthesis. Primary structure deduced from the cDNA sequence  

Microsoft Academic Search

We carried out the cloning of a mouse cDNA encoding a sepiapterin reductase which is involved in the final step of tetrahydrobiopterin biosynthesis as a first step toward gene-targeting technique in mice. The sequence contained 1245 nucleotides consisting of an open reading frame of 783 nucleotides encoding a protein of 261 amino acid residues whose molecular weight was 27851, a

Akira Ota; Hiroshi Ichinose; Toshiharu Nagatsu

1995-01-01

303

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 80°C for 30 min and analyzed by GC-MS. Linearity ranged from 10 to 500 ng/mL, except for ecgonine methyl ester, for which linearity ranged from 10 to 100 ng/mL. Inter- and intra-day imprecision ranged from 2.8 to 18.4% and from 1.5 to 14.9%, respectively. Accuracy values lay between 86.9 and 104.4%. The limit of quantitation for all drugs was 10 ng/mL and recoveries were >74% for all analytes, except for cannabinoids, which showed poor recovery (?30%). The developed method was applied to real samples collected from deceased victims due to traffic accidents. These samples were selected according to the results obtained in immunoassay screening on collected urine samples. Five samples were positive for the presence of COC and metabolites, four samples were positive for cannabinoids, six samples were positive for AMPs and two samples were drug negative. Some samples were positive for more than one class of drug. Results obtained from whole blood samples showed good agreement with urine screening. The developed method proved capable of quantifying all three classes of drugs of abuse proposed in this study, through a one-step extraction procedure. PMID:24782143

Pelição, Fabrício Souza; Peres, Mariana Dadalto; Pissinate, Jauber Fornaciari; De Martinis, Bruno Spinosa

2014-07-01

304

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

PubMed Central

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

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

2011-01-01

305

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

PubMed

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

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

2008-04-01

306

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.

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

2011-01-01

307

Down-Regulation of DNA Replication in Extracts of Camptothecintreated Cells: Activation of an S-phase Checkpoint?1  

Microsoft Academic Search

Extracts prepared from camptothecin(CPT)-treatedcells have a re duced ability to support SV4ODNA replication in vitro. This reduction derives mainly from a reduction in the frequency of initiation events because DNA chain elongation remains practically unchanged. Mixing of extract from nontreated cells with small amounts of extract of CPT treated cells indicates that the reduction In DNA replication is due to

Ya Wang; Ange Ronel Perrault; George Hiakis

308

Extraction of DNA from Serum for High-throughput Genotyping: Findings from Pilot Studies within the Prostate Cancer Prevention Trial  

PubMed Central

OBJECTIVES DNA extraction from blood and genotyping for candidate single nucleotide polymorphisms (SNP) is now an important part of almost all molecular epidemiologic studies. However, in many studies, the amount of blood sample is limited or only serum is available. We conducted several pilot studies to identify methods for DNA extraction and high-throughput SNP genotyping of both white blood cell (WBC) and serum DNA that can be done centrally and reliably for large numbers of samples. METHODS We used biospecimens from The Prostate Cancer Prevention Trial (PCPT), a phase III, double-blind, placebo-controlled trial that tested the efficacy of finasteride for the primary prevention of prostate cancer. DNA was extracted from WBCs, from serum, and also from serum after organic solvent extraction for analysis of hormones. We also conducted blinded high-throughput genotyping in 3 laboratories to assess feasibility and reliability of results with differing methodologies using DNA from WBCs and from serum. RESULTS Genotyping of DNA extracted from WBCs resulted in highly reliable, reproducible results across laboratories using different genotyping platforms. However, genotyping with DNA extracted from serum did not provide reliable data using high-throughput multiplex approaches such as Sequenom (hME and iPLEX) and Applied Biosystems SNPlex, but was successful using Taqman. CONCLUSIONS Based upon the results of these pilot studies, we conclude that DNA obtained from serum must be used judiciously, and that genotyping using multiplex methods is not suitable for serum DNA.

Hoque, Ashraful; Goodman, Phyllis; Ambrosone, Christine B.; Figg, William D.; Price, Doug; Koop, William; Wu, Xifeng; Conroy, Jeffrey; Lehman, Teresa A.; Santella, Regina M.

2008-01-01

309

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

PubMed Central

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

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

2011-01-01

310

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

PubMed

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

Demeke, Tigst; Jenkins, G Ronald

2010-03-01

311

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

SciTech Connect

The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin`s lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. 30 refs., 7 figs.

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

1995-06-01

312

An effective method of DNA extraction for bioleaching bacteria from acid mine drainage.  

PubMed

An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6-10 min, followed by 60 +/- 5 degrees C for 30 min, then 72 degrees C for 30 min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies. PMID:18481056

Zeng, Leping; Huang, Jufang; Zhang, Yanfei; Qiu, Guanzhou; Tong, Jianbin; Chen, Dan; Zhou, Jin; Luo, Xuegang

2008-07-01

313

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.

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

2012-01-01

314

DNA extraction and amplification of 10-day, room-temperature blood samples.  

PubMed

DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation. PMID:10730541

Sasanakul, W; Chuansumrit, A; Rurgkhum, S; Udomsubpayakul, U; Hathirat, P

1999-11-01

315

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

PubMed

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

2013-11-01

316

Astrocyte activation: a key step in rotenone induced cytotoxicity and DNA damage.  

PubMed

Astrocytes are the most abundant glial cells, which provide metabolic support for neurons. Rotenone is a botanical pesticide of natural origin, known to exhibit neurotoxic potential via inhibition of mitochondrial complex-I. This study was carried out to explore the effect of rotenone on C6 cells. The cell line C6 derived from rat glioma cells represents astrocyte-like cell. C6 cells were treated with rotenone (0.1, 1 and 10 ?M) for 4 h. The effect of rotenone was studied on cell survival (MTT reduction and PI uptake); free radicals (ROS and RNS) and DNA damage (comet assay and Hoechst staining). The glial cell activation and apoptotic cell death was evaluated by expression of Glial fibrillary acidic protein (GFAP) and caspase-3 respectively. The treatment with rotenone resulted in decreased cell survival and increased free radical generation. Altered nuclear morphology and DNA damage were evident following rotenone treatment in Hoechst staining and Comet assay. Rotenone elevated expression of GFAP and caspase-3 that indicates glial cell activation and apoptosis, respectively. We further studied the effect of melatonin, an antioxidant, on the observed toxic effects. Co-incubation of antioxidant, melatonin (300 ?M), significantly suppressed rotenone induced above-mentioned effects in C6 cells. Inhibitory effects of melatonin suggest that free radicals play a major role in rotenone induced astrocyte activation and cellular toxicity leading to apoptosis of astroglial cells. PMID:22846965

Swarnkar, Supriya; Singh, Sarika; Goswami, Poonam; Mathur, Ramesh; Patro, Ishan K; Nath, Chandishwar

2012-10-01

317

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

PubMed Central

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

2012-01-01

318

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.

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

2014-01-01

319

One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex.  

PubMed

Though opening of the start site (+1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisingly little is known about how and when this occurs in the mechanism. Early events at the lambdaP(R) promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I(1). Conversion to the subsequent intermediate I(2) overcomes a large enthalpic barrier. Is I(2) open? Here we create a burst of I(2) by rapidly destabilizing open complexes (RP(o)) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I(2) are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I(2) to I(1) conversion rate determined by filter binding. In I(2), permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RP(o) control, whereas nontemplate (nt) thymines are significantly less reactive than in RP(o). We propose that: (i) the +1(t) thymine is in the active site in I(2); (ii) conversion of I(2) to RP(o) repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RP(o). We hypothesize that unstable open intermediates at the lambdaP(R) promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters. PMID:20483995

Gries, Theodore J; Kontur, Wayne S; Capp, Michael W; Saecker, Ruth M; Record, M Thomas

2010-06-01

320

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

NASA Astrophysics Data System (ADS)

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

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

2012-01-01

321

A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR  

Microsoft Academic Search

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described. Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.

Joe Attal; Marco Cajero-Juarez; Louis-Marie Houdebine

1995-01-01

322

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

323

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

324

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

PubMed Central

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

2014-01-01

325

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

PubMed

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

Sirdah, Mahmoud Mohammed

2014-04-01

326

Long PCR-based Genotyping for a Deleted CYP2D6 Gene without DNA Extraction.  

PubMed

In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand. Cytochrome P450 (CYP) 2D6 is one of the most widely investigated CYPs in relation to genetic polymorphism. Detection of CYP2D6*5 is difficult since long PCR is used. Especially for samples without DNA extraction, the detection is not sensitive enough for population analysis. Therefore, we developed a CYP2D6*5 genotyping method that involves nested long PCR, directly using human whole saliva as a template without DNA extraction. This method will be very useful for genetic diagnoses and can be an efficient tool for individualization of drug therapy in clinical studies. PMID:24390472

Ota, Tomoko; Hayashida, Mariko; Ishii, Minori; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

2014-06-25

327

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens  

PubMed Central

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, ?-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the ?-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAFV600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used.

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

2013-01-01

328

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

PubMed

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

Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

2008-12-01

329

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

PubMed

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

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

1991-09-01

330

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

PubMed

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

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

2004-11-17

331

Cereal DNA: A rapid high-throughput extraction method for marker assisted selection  

Microsoft Academic Search

A rapid, automated and novel method is presented to extract DNA suitable for polymerase chain reaction (PCR) from small amounts\\u000a of cereal leaf tissue in a high-throughput cost-effective way. The method uses a 96-well plate in which leaf samples are frozen,\\u000a mechanically crushed using a matrix mill, macerated in alkali and subsequently neutralized. The method was used routinely\\u000a with barley

Maxime Paris; Meredith Carter

2000-01-01

332

Semiconservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template1  

Microsoft Academic Search

We describe the preparation of nuclear extracts from yeast cells synchro- nised 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 intermedi- ates that have incorporated (a- 32 P)dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by

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

1998-01-01

333

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

Microsoft Academic Search

The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in

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

2004-01-01

334

Microsatellite markers to identify specific alleles in DNA extracted from monovarietal virgin olive oils  

Microsoft Academic Search

The suitability of DNA present in olive oil for PCR analysis has been reported by several authors. However, low concentration,\\u000a degradation, and the possible presence of additional alleles due to paternal contribution in oils extracted from entire drupes,\\u000a should be taken into consideration when comparing the amplification profiles of leaves with the corresponding oils for varietal\\u000a traceability purposes. The aim

Vittorio Alba; Wilma Sabetta; Antonio Blanco; Antonella Pasqualone; Cinzia Montemurro

2009-01-01

335

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.

Ronayne, Erin A.; Cox, Michael M.

2014-01-01

336

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

PubMed

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

Ronayne, Erin A; Cox, Michael M

2014-04-01

337

Annotating genes and genomes with DNA sequences extracted from biomedical articles  

PubMed Central

Motivation: Increasing rates of publication and DNA sequencing make the problem of finding relevant articles for a particular gene or genomic region more challenging than ever. Existing text-mining approaches focus on finding gene names or identifiers in English text. These are often not unique and do not identify the exact genomic location of a study. Results: Here, we report the results of a novel text-mining approach that extracts DNA sequences from biomedical articles and automatically maps them to genomic databases. We find that ?20% of open access articles in PubMed central (PMC) have extractable DNA sequences that can be accurately mapped to the correct gene (91%) and genome (96%). We illustrate the utility of data extracted by text2genome from more than 150 000 PMC articles for the interpretation of ChIP-seq data and the design of quantitative reverse transcriptase (RT)-PCR experiments. Conclusion: Our approach links articles to genes and organisms without relying on gene names or identifiers. It also produces genome annotation tracks of the biomedical literature, thereby allowing researchers to use the power of modern genome browsers to access and analyze publications in the context of genomic data. Availability and implementation: Source code is available under a BSD license from http://sourceforge.net/projects/text2genome/ and results can be browsed and downloaded at http://text2genome.org. Contact: maximilianh@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

Haeussler, Maximilian; Gerner, Martin; Bergman, Casey M.

2011-01-01

338

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

PubMed

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

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

2014-08-01

339

Single-molecule analysis of DNA replication in Xenopus egg extracts.  

PubMed

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface. PMID:22503776

Yardimci, Hasan; Loveland, Anna B; van Oijen, Antoine M; Walter, Johannes C

2012-06-01

340

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

PubMed

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

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

2012-01-01

341

Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing.  

PubMed

Body fluid identification and individual identification are an important series of tests in usual criminal investigations. Recent reports have demonstrated a new approach using DNA/RNA co-extraction methods in which RNA for body fluid identification and DNA for short tandem repeat (STR) typing are extracted simultaneously from the same sample. This study evaluated a standard co-extraction kit, the AllPrep® DNA/RNA Mini Kit, in order to demonstrate the availability of the co-extraction procedure for those real-time polymerase chain reaction-based body fluid identification methods that we have validated previously. We demonstrated that the use of the Allprep Kit, for which we adjusted the lysis temperature to 56°C to improve extraction efficiency, can simultaneously extract sufficient RNA and DNA for body fluid identification and STR analysis; however, a longer incubation at a high temperature slightly affected the ?Ct value of each target gene and appeared to be not as effective for DNA extraction from old stains as from 1-day-old stains. This method is promising for future forensic investigations because the use of this kit can reduce sample consumption for body fluid identification and DNA typing. PMID:24296038

Watanabe, Ken; Iwashima, Yasuki; Akutsu, Tomoko; Sekiguchi, Kazumasa; Sakurada, Koichi

2014-01-01

342

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

PubMed

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

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

2014-03-01

343

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

PubMed Central

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

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

2012-01-01

344

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.  

PubMed

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself. PMID:9891057

Duncker, B P; Pasero, P; Braguglia, D; Heun, P; Weinreich, M; Gasser, S M

1999-02-01

345

Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts.  

PubMed

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) has a well-established role in epigenetic regulation through the recognition of various histone marks and interaction with chromatin-modifying proteins. However, its function in regulating cell cycle progression remains poorly understood and has been largely attributed to a role in transcriptional regulation. In this study we have used Xenopus laevis egg extracts to analyse Uhrf1 function in DNA replication in the absence of transcriptional influences. We demonstrate that removal of Uhrf1 inhibits chromosomal replication in this system. We further show that this requirement for Uhrf1, or an associated factor, occurs at an early stage of DNA replication and that the consequences of Uhrf1 depletion are not solely due to its role in loading Dnmt1 onto newly replicated DNA. We describe the pattern of Uhrf1 chromatin association before the initiation of DNA replication and show that this reflects functional requirements both before and after origin licensing. Our data demonstrate that the removal of Xenopus Uhrf1 influences the chromatin association of key replication proteins and reveal Uhrf1 as an important new factor required for metazoan DNA replication. PMID:23788677

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

2013-09-01

346

Simultaneous extraction of proteins and DNA by an enzymatic treatment of the cell wall of Palmaria palmata (Rhodophyta)  

Microsoft Academic Search

The extraction of proteins and DNA from seaweed tissue is difficult due to the presence of cell wall anionic polysaccharides\\u000a that, after cell disruption, remain in the extraction medium as hydrocolloidal compounds. These compounds increase medium\\u000a viscosity, thus limiting access to and, consequently, quantification of the soluble macromolecules such as proteins or DNA.\\u000a This study describes a protocol that enables

Yolaine Joubert; Joël Fleurence

2008-01-01

347

Fast wheat variety classification by capillary gel electrophoresis-on-a-chip after single-step one-grain high molecular weight glutenin extraction.  

PubMed

Wheat variety identification based on one-step single-grain wheat extraction and fast capillary gel electrophoresis-on-a-chip (CGE-on-a-chip) analyses was evaluated for 15 different wheat varieties grown in Austria. The results of the capillary-based separation system were compared to the internationally accepted method from the International Union for the Protection of New Varieties of Plants which is based on time-consuming sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparable protein patterns were observed making the CGE-on-a-chip system a promising tool for high-throughput analysis in food control. For the development of a robust method protein extraction, shelf life of wheat extracts and the instrument's variability were evaluated. It turned out that a one-step single-grain wheat extraction allowed the sample to be stored at 4 °C for up to 4 weeks without losing any valuable protein information. Furthermore, the technical variation of the whole method is very low making the biological variation of the selected wheat grains the only uncertain factor. Additionally, two unsupervised statistical methods (hierarchical cluster analysis and principal component analysis) were used for variety identification. Identification was successful for a reduced data set of 14 samples from five different wheat varieties making the combination of CGE-on-a-chip analysis of one-step single-grain extraction in combination with automatic data evaluation a promising tool for fast wheat differentiation (within a day). PMID:21298418

Marchetti-Deschmann, Martina; Lehner, Angela; Peterseil, Verena; Sövegjarto, Friedrich; Hochegger, Rupert; Allmaier, Günter

2011-06-01

348

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

PubMed Central

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

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

2010-01-01

349

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.  

PubMed

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; Hémon, 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; Sánchez, Maria-José; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Núria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre

2012-01-01

350

An HPTLC method for the determination of minocycline in human plasma, saliva, and gingival fluid after single step liquid extraction.  

PubMed

A rapid, sensitive and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for determination of minocycline in human plasma, saliva, and gingival fluid samples. Densitometric analysis of minocycline was carried out at 345 nm after single step extraction with methanol. The method uses TLC aluminium plates pre-coated with silica gel 60F-254 as a stationary phase and methanol-acetonitrile-isopropyl alcohol-water (5:4:0.5:0.5, v/v/v/v) as mobile phase. In all the three matrices, the calibration curve was linear (r(2) >/= 0.9958) in the tested range of 100 - 1200 ng spot(-1) with a limit of quantification of 15.4 ng spot(-1). Drug recovery from plasma, saliva and gingival fluid averaged 97.7%. Intra- and inter-day accuracies, determined at three different concentrations, were 95.08 to 100.6% and the corresponding precision (% CV) values were < 4.61%. In all the three matrices, rapid degradation of drug occurred and the half-life of drug ranged from 9.9 to 16.1 h at 4 degrees C and from 6.3 to 11.5 h at 20 degrees C. Frozen at -20 degrees C, this drug was stable for at least 2 months and can tolerate two freeze-thaw cycles without losses higher than 10%. The method's ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies. PMID:19139573

Jain, Nilu; Jain, Gaurav Kumar; Iqbal, Zeenat; Talegaonkar, Sushma; Ahmad, Farhan Jalees; Khar, Roop Krishen

2009-01-01

351

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/µl). The same 13 clinical samples (13/31; 42%) were positive using both DNA extraction methods with the lowest limits of detection. Conclusions Simple and low-cost methods can be sensitive and useful in extracting DNA from DBS. Poor results on stored clinical DBS indicate that further studies on the impact of storage duration and conditions, and choice of filter paper should be performed.

Str?m, Gro E. A.; Tellevik, Marit G.; Hanevik, Kurt; Langeland, Nina; Blomberg, Bj?rn

2014-01-01

352

Evaluation of DNA extraction methods and dilution treatment for detection and quantification of Acanthamoeba in water and biofilm by real-time PCR.  

PubMed

Acanthamoeba, human pathogens and natural hosts of pathogenic bacteria, may be accurately detected and quantified by real-time PCR if Acanthamoeba DNA are properly extracted and PCR inhibitors are effectively eliminated. However, the optimization of DNA extraction methods has not been reported for Acanthamoeba. This study compared the effectiveness of two DNA extraction/purification methods (FastDNA(®) Spin Kit for soil and Wizard(®) SV genomic DNA Purification System) by using trophozoites and cysts of Acanthamoeba castellanii and water and biofilm samples of cooling towers. DNA of A. castellanii extracted with the FastDNA(®) Kit and quantified by TaqMan PCR resulted in a lower variation (CV of Ct < 3%), greater linearity (R(2)=0.99), and higher slopes (1.177-1.187 log fg DNA/log cell number) as compared to that by the Wizard(®) Kit. For field testing, the number of Acanthamoeba-positive samples and the Acanthamoeba DNA quantity were both greater with the FastDNA(®) Kit than with the Wizard(®) Kit (P=0.016 and <0.0001, respectively). Beneficial effects with dilutions of extracted DNA were also revealed with the FastDNA(®) Kit (P=0.0003). In conclusion, DNA extraction by the FastDNA(®) Kit coupled with dilution of extracted DNA and PCR analysis are recommended for detecting and quantifying environmental Acanthamoeba. PMID:21045343

Chang, Ching-Wen; Wu, Ying-Chieh

2010-01-01

353

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

354

What do results of common sequential fractionation and single-step extractions tell us about P binding with Fe and Al compounds in non-calcareous sediments?  

PubMed

Correct identification of P forms together with their main Fe and Al binding partners in non-calcareous sediments is of crucial importance for evaluation of P cycling in water bodies. In this paper, we assess extraction methods frequently used for this purpose, i.e., a sequential five-step fractionation (water, bicarbonate buffered dithionite solution (BD), NaOH, HCl, nitric-perchloric acid), ascorbate extraction (pH ~7.5), and oxalate extraction (pH ~3), directly on a range of laboratory prepared Fe and Al minerals enriched with adsorbed P. Extraction selectivity and efficiency for particular P, Fe and Al forms were also verified by specific combinations of these extraction methods applied on freshwater sediment samples. In the sequential fractionation, BD was highly effective in dissolving both amorphous and crystalline Fe (hydr)oxides and the associated P, while neither FeS nor Al (hydr)oxides were dissolved. The following NaOH extraction effectively dissolved both amorphous and crystalline Al (hydr)oxides. The high solubilizing power of BD and NaOH to dissolve crystalline Fe and Al oxides that have only a small P-sorption ability prevents the use of resulting Fe/P and Al/P ratios as simple predictors of total P sorption capacity of sediments and soils. Ascorbate non-selectively extracted small proportions of FeS and amorphous Fe and Al (hydr)oxides, but significant amounts of adsorbed P, which hinders its use for the characterization of P forms in non-calcareous sediments. Similar nonselective characteristics were found for oxalate extractions. As oxalate extracts most of the adsorbed phosphate, it is not possible to use it unambiguously to determine specific Fe/P and Al/P ratios of active complexes. However, this method is convenient (and more selective than NaOH step in the sequential fractionation) for the determination of amorphous Al (hydr)oxides. PMID:23218245

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

2013-02-01

355

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

356

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

PubMed Central

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (108 and 107 CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 108 CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P ? 0.005), 107 CFU of C. neoformans (P ? 0.05), and 107 CFU of A. fumigatus (P ? 0.01). Yields were within the same range for 108 CFU of C. neoformans and 107 CFU of C. albicans for both HSCD extraction and PC extraction. For 107 CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P ? 0.05). Yields obtained from 108 and 107 CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

Muller, Frank-Michael C.; Werner, Katherine E.; Kasai, Miki; Francesconi, Andrea; Chanock, Stephen J.; Walsh, Thomas J.

1998-01-01

357

Identification of a Preinitiation Step in DNA Replication That Is Independent of Origin Recognition Complex and cdc6, but Dependent on cdk2  

Microsoft Academic Search

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and mini- chromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation com- plexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initi- ation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic

Xuequn Helen Hua; John Newport

1998-01-01

358

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

Microsoft Academic Search

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3)

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

2009-01-01

359

Preparation of magnetite-loaded silica microspheres for solid-phase extraction of genomic DNA from soy-based foodstuffs  

Microsoft Academic Search

Solid-phase extraction has been widely employed for the preparation of DNA templates for polymerase chain reaction (PCR)-based analytical methods. Among the variety of adsorbents studied, magnetically responsive silica particles are particularly attractive due to their potential to simplify, expedite, and automate the extraction process. Here we report a facile method for the preparation of such magnetic particles, which entails impregnation

Ruobing Shi; Yucong Wang; Yunli Hu; Lei Chen; Qian-Hong Wan

2009-01-01

360

A DNA extraction procedure that allows mite specimens to be slide mounted: phytoseiid species evaluated as a model.  

PubMed

Four protocols for extracting DNA from mites, using phytoseiid species as exemplars, were evaluated to determine whether the DNA obtained could be used to amplify nuclear, mitochondrial or Random Amplified Polymorphic DNA (RAPD) markers from males, females and eggs. Protocol 3 was identified as the best and this allowed High-fidelity PCR (Hf-PCR) and Hf-RAPD PCR to be used successfully; it left behind the intact body of adult mites so they could be slide mounted for morphological analyses, although the eggs had to be pricked in order to yield sufficient DNA for amplifications. Protocol 3 involved soaking intact specimens in a GuSCN buffer and using a silica matrix, which binds nucleic acids, to yield DNA for amplification. The DNA isolated could be stored up to a month, indicating that the quality was good. This DNA extraction protocol will allow researchers to collect mites, store them in 95% ethanol, and subsequently extract sufficient DNA from single adults or eggs to provide diagnostic PCR products from both nuclear and mitochondrial DNA, yet leave the bodies intact for morphological analyses. PMID:20333447

Jeyaprakash, Ayyamperumal; Hoy, Marjorie A

2010-10-01

361

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical.  

PubMed

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS(+) assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts. PMID:21570443

Chatti, Ines Bouhlel; Boubaker, Jihed; Skandrani, Ines; Bhouri, Wissem; Ghedira, Kamel; Chekir Ghedira, Leila

2011-08-01

362

Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition  

PubMed Central

Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g?1 using Griffiths and 4.25×106 cells g?1 using FastDNA® Spin kit.

Pontiroli, Alessandra; Travis, Emma Rachel; Sweeney, Francis Patrick; Porter, David; Gaze, William Hugo; Mason, Sam; Hibberd, Victoria; Holden, Jennifer; Courtenay, Orin; Wellington, Elizabeth Margaret Helen

2011-01-01

363

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.

2008-03-01

364

DNA Extraction from Dry Museum Beetles without Conferring External Morphological Damage  

PubMed Central

Background A large number of dry-preserved insect specimens exist in collections around the world that might be useful for genetic analyses. However, until now, the recovery of nucleic acids from such specimens has involved at least the partial destruction of the specimen. This is clearly undesirable when dealing with rare species or otherwise important specimens, such as type specimens. Methodology We describe a method for the extraction of PCR-amplifiable mitochondrial and nuclear DNA from dry insects without causing external morphological damage. Using PCR to amplify ?220 bp of the mitochondrial gene cytochrome c oxidase I, and 250–345 bp fragments of the multi-copy, nuclear 28s ribosomal DNA gene, we demonstrate the efficacy of this method on beetles collected up to 50 years ago. Conclusions This method offers a means of obtaining useful genetic information from rare insects without conferring external morphological damage.

Gilbert, M. Thomas P.; Moore, Wendy; Melchior, Linea; Worobey, Michael

2007-01-01

365

Extract from Agaricus blazei Murill can enhance immune responses elicited by DNA vaccine against foot-and-mouth disease.  

PubMed

The fungus Agaricus blazei Murill (ABM) is particularly rich in polysaccharides, which have shown particularly strong results in treating and preventing cancers. The goal of this study was to investigate whether co-administration of the ABM extract with foot-and-mouth disease virus (FMDV) DNA vaccine could increase the immune responses. Compared with the control mice, which received FMDV DNA vaccine alone, significant increase in not only the FMDV-specific antibody response but also T cell proliferation was observed in mice which received FMDV DNA vaccine plus the ABM extract. Taken together, these results demonstrated that application of the ABM extract might provide a strategy to improve the efficacy of DNA vaccines. PMID:16213597

Chen, Liang; Shao, Hanjuan

2006-01-15

366

Determination of O6-butylguanine in DNA by immunoaffinity extraction\\/gas chromatography-mass spectrometry  

Microsoft Academic Search

A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its

Marina Bonfanti; Cinzia Magagnotti; Alessandra Galli; Renzo Bagnati; Massimo Moret; Pierluigi Gariboldi; Roberto Fanelli; Luisa Airoldi

1990-01-01

367

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

PubMed

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device. PMID:23370016

Oblath, Emily A; Henley, W Hampton; Alarie, Jean Pierre; Ramsey, J Michael

2013-04-01

368

A Simple and Accurate Two-Step Long DNA Sequences Synthesis Strategy to Improve Heterologous Gene Expression in Pichia  

PubMed Central

In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200–500 bp fragments with 20–25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

Yang, Jiang-Ke; Chen, Fang-Yuan; Yan, Xiang-Xiang; Miao, Li-Hong; Dai, Jiang-Hong

2012-01-01

369

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

PubMed

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

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

1993-09-01

370

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

PubMed

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

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

2006-06-01

371

Technical note: Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods  

Microsoft Academic Search

Several new automated methods have recently become available for high-throughput DNA extraction, including the Maxwell 16 System (Promega UK, Southampton, UK). The purpose of this report is to compare automated with manual DNA extraction methods, and invasive with noninvasive sample collection methods, in terms of DNA yield and quality. Milk, blood, and nasal swab samples were taken from 10 cows

C. Foley; C. O’Farrelly; K. G. Meade

2011-01-01

372

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 49g/L. Hydrothermal treatment (180°C - 20min) 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, Ozge; Reisinger, Michael; Huber, Florian; Kneifel, Wolfgang; Novalin, Senad

2014-07-01

373

Evaluation of DNA Extraction Techniques for Detecting Mycobacterium tuberculosis Complex Organisms in Asian Elephant Trunk Wash Samples?  

PubMed Central

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

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

2011-01-01

374

The joining of non-complementary DNA double-strand breaks by mammalian extracts.  

PubMed Central

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini. This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from blunt-end joining by the biochemical fractionation of extracts. Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.

Mason, R M; Thacker, J; Fairman, M P

1996-01-01

375

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

376

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

PubMed Central

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

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

2008-01-01

377

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

378

PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.  

PubMed

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary. PMID:15322425

Byers, Richard; Roebuck, Jamie; Sakhinia, Ebrahim; Hoyland, Judith

2004-09-01

379

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

PubMed

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

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

2010-01-01

380

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

PubMed Central

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

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

381

EXTRAÇÃO NÃO-INVASIVA DE DNA DE \\  

Microsoft Academic Search

Molecular genetics has been a powerful aid in animal breeding. DNA extraction is an important step and can be done by several methods, including some noninvasive ones, more attractive in custs and easiness of execution. It was compare five DNA extraction methods applied to samples of hair shafts and oral mucosa cells (swab). Those methods based in Chelex 100 performed

GUSTAVO AUGUSTO LACORTE; MARIA RAQUEL SANTOS

382

Development of a one-step integrated pressurized liquid extraction and cleanup method for determining polycyclic aromatic hydrocarbons in marine sediments.  

PubMed

A rapid and accurate one-step integrated pressurized liquid extraction (PLE) and cleanup method was developed and validated for 34 polycyclic aromatic hydrocarbons in marine sediments, giving an extract that could be analyzed by gas chromatography-mass spectrometry without further cleanup. Marine sediment (5g) was loaded into the stainless-steel extraction cell above activated copper (5g) and activated silica gel (5g). An extraction temperature of 100°C and two 5min extraction cycles using a 4:1 (v/v) hexane-dichloromethane mixture gave a good extraction efficiency. The integrated method gave extracts that were as clean as those obtained using PLE, followed by separate activated copper and silica gel cleanups. The method was validated, in terms of its accuracy, precision, and application using a certified reference material (NIST SRM 1944), marine sediments spiked at low and high concentrations, and contaminated harbor sediments. The mean recoveries were 92% and 94% for the low and high spike concentrations, respectively, and the accuracy was good (giving a mean of 86% of the certified reference material concentrations). The method developed gave a precision and accuracy equal to or better than the precision and accuracy found using PLE with separate cleanups. The method developed gives a shorter sample preparation time and uses much less solvent than PLE and separate cleanups. PMID:24671040

Choi, Minkyu; Kim, Ye-Jung; Lee, In-Seok; Choi, Hee-Gu

2014-05-01

383

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control  

Microsoft Academic Search

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of

M. M. Klerks; Bruggen van A. H. C; C. Zijlstra; M. Donnikov; Vos de R

2006-01-01

384

Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.  

PubMed Central

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.

Thomas, D C; Svoboda, D L; Vos, J M; Kunkel, T A

1996-01-01

385

Comparative evaluation of Amplicor HIV-1 DNA test, version 1.5, by manual and automated DNA extraction methods using venous blood and dried blood spots for HIV-1 DNA PCR testing.  

PubMed

Human immunodeficiency virus (HIV) DNA polymerase chain reaction (PCR) test using venous blood sample has been used for many years in low resource settings for early infant diagnosis of HIV infection in children less than 18 months. The aim of this study was to evaluate and compare the performance characteristics of Amplicor HIV-1 DNA assay version 1.5 following processing of venous blood and dried blood spot (DBS) samples by Roche manual DNA extraction and automated Roche MagNA Pure LC instrument (MP) for HIV-1 DNA PCR testing in Dar es Salaam, Tanzania, in order to scale up early infant diagnosis of HIV infection in routine practice. Venous blood samples from children under 18 months born to HIV-infected mothers between January and April 2008 were collected. Venous blood was used to prepare cell pellet and DBS samples. DNA extractions by manual procedure and MP were performed each on cell pellet, venous blood and DBS samples and tested by Amplicor HIV-1 DNA assay. Of 325 samples included, 60 (18.5%) were confirmed HIV-infected by manual extraction performed on cell pellets. Sensitivity of the assay following MP processing of venous blood was 95% (95% CI; 86.1-99.0%) and 98.3% (95% CI; 91.1 to 99.9%) for the manual extraction and processing by MP performed on DBS samples. Specificity of the assay with all DNA extraction methods was 99.6% (95% CI; 97.9 to 100%). Performance of the assay with Roche manual extraction and processing by MP on DBS samples compared well with Roche manual extraction performed on cell pellet samples. The choice of DNA extraction method needs to be individualized based on the level of laboratory facility, volume of testing and cost benefit analysis before it is adopted for use. PMID:24409629

Nsojo, Anthony; Aboud, Said; Lyamuya, Eligius

2010-10-01

386

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

387

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability  

PubMed Central

Background The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Methods DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. Results The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. Conclusions The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

2013-01-01

388

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

PubMed

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF(103) of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used. PMID:19922433

Hjort, Karin; Bergström, Maria; Adesina, Modupe F; Jansson, Janet K; Smalla, Kornelia; Sjöling, Sara

2010-02-01

389

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

PubMed

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

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

2008-12-10

390

NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis  

Microsoft Academic Search

One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and

Yi-Chun Wu; Gillian M. Stanfield; H. Robert Horvitz

2000-01-01

391

Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry  

SciTech Connect

A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.

Bonfanti, M.; Magagnotti, C.; Galli, A.; Bagnati, R.; Moret, M.; Gariboldi, P.; Fanelli, R.; Airoldi, L. (Istituto di Ricerche Farmacologiche, Mario Negri, Milan (Italy))

1990-11-01