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At least 235 records · Page 13Linked to original sources
PubMed2005

Comparison of five DNA extraction methods and optimization of a b1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain.

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide. Different techniques have been developed for T. gondii detection. At present, polymerase chain reaction (PCR) has been widely used. However, PCR for identifying T. gondii remains unsatisfactory in many laboratories because of lack of standardization and variations in efficiency. In the present study, we optimized a nested PCR protocol (n-PCR) in order to compare the amplification of T. gondii DNA, after being extracted from mouse brain by five different DNA extraction methods including phenol chloroform, QIAamp DNA minikit, Genomic DNA purification kit and Chelex with or without proteinase K. All DNA extraction methods were able to extract DNA from a single tissue cyst from mouse brain. However, among the five DNA extraction methods, the Chelex without proteinase K appeared to be the most rapid and easiest.

Animals↗
PubMed2001

A strategy for optimizing quality and quantity of DNA extracted from soil.

The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.

Buffers↗
PubMed2006

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

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

Automation↗
PubMed1998

DNA extraction from human urinary sediment.

DNA was extracted from urinary sediments and was sufficient for polymerase chain reaction (PCR) and enzymatic analysis, even if DNA from microorganisms coexisted. From urine samples, the yield of DNA ranged from trace levels to 20 micrograms per 10 mL urine. When urinary sediment was stored in ethanol, DNA remained stable for 2 weeks or more. Individual identification and sex determination could easily be performed using either fresh or ethanol-fixed urine. In conclusion, urine can be used as a source for PCR-based investigations and genetic studies.

Adolescent↗
PubMed2004

A simple and efficient method for extracting DNA from old and burned bone.

It has been a challenge to extract DNA from bones previously soaked in water, burned, or buried for a long time, due to the reduced quality and quantity of DNA in the bone samples. The dramatic degradation of the DNA and the presence of PCR inhibitors in the collagen significantly complicate the process of DNA identification in dated and charred bones. In this article, we present a novel strategy to obtain DNA from bones based on the use of cetyltrimethylammonium bromide (CTAB) lysis buffer and isoamyl alcohol-chloroform extraction with subsequent DNA purification using the DNA IQ System, or alternatively the QIAquick system. When applied to bones soaked, burned or buried for up to nine years, this method increases the purity and yield of DNA with respect to the traditional phenol-chloroform method and significantly improves multiplex STR genotyping using fluorescence-based methods. The results of this research will assist forensic scientists in the identification of DNA from victims whose bodies underwent significant trauma or burning, precluding the utilization of traditional forensic DNA identification techniques.

Bone and Bones↗
PubMed2004

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

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.

Animals↗
PubMed2004

One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.

The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.

Animals↗
PubMed1981

DNA extraction during Giemsa differentiation of chromatids singly and doubly substituted with BrdU.

Human lymphocytes were cultured in 3H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsa-stained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls.--The differentiation method involved sequential pretreatments with short wave UV and 2 x SSc at 60 degrees C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly.--On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of 3H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label.--Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.

Autoradiography↗
PubMed1997

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

Using specimens spiked with BK virus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from urine samples. It was found that PCR inhibition could be completely overcome by extracting samples with 30% polyethylene glycol (PEG) in 3 M sodium chloride, and partially overcome by extracting samples with guanidine thiocyanate in the presence of high salt concentrations. The nature of the sample inhibition was investigated, leading to the conclusion that both urea and unidentified non-proteinaceous DNA associated substances inhibit BKV DNA amplification from urine.

BK Virus↗
PubMed1986

Transforming activity of DNA extracted from human urological tumours.

The transforming activity of DNA extracted from one testicular cancer, one benign prostatic hyperplasia, two malignant prostatic adenocarcinomas, six renal cell carcinomas, two tumours of the renal pelvis, a specimen of normal urothelial tissue, two benign papillomas of the bladder and five malignant bladder carcinomas, was investigated using a DNA transfection assay on primary cultures of hamster kidney cells (HK cells) and mouse embryo fibroblasts (MEF cells). Transformation was scored under light microscope by the appearance of foci of morphologically transformed cells and by evaluating the ability of the transfected cells to from colonies in soft agar. Primary HK cells were unable to support transformation by human tumour DNA because of their very limited lifespan in vitro; MEF cultures, on the contrary, had a much more prolonged life: but were only abortively transformed by DNA from four out of six renal cell carcinomas and four out of seven malignant urothelial tumours, suggesting that transforming genes are present but not activated in the DNA from these urological malignancies.

Animals↗
PubMed2003

Y-chromosomal short tandem repeats haplotyping from vaginal swabs using a chelating resin-based DNA extraction method and a dual-round polymerase chain reaction.

Reported are 2 autopsy cases in which Y-chromosomal microsatellite short tandem repeats DYS19, DYS389I and II, DYS390, and DYS393 could be haplotyped with vaginal swabs by using a Chelex 100-based DNA extraction method and dual-round polymerase chain reaction. The extraction of DNA from vaginal swabs by using this method was as efficient or more efficient than using proteinase K and phenol-chloroform extraction or the alkaline lysis methods. Y-chromosomal microsatellite short tandem repeats haplotyping based on the dual-round polymerase chain reaction method provided genotypes from all the loci determined. Although amplification of Y-chromosomal microsatellite short tandem repeats loci is not directly involved in the existence of spermatozoa, it is considerably advantageous for male individualization from body fluid mixture stains in criminal cases.

Adult↗
PubMed2005

Short report: Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum.

The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive, and cost-effective DNA extraction methods. We describe a new Tris-EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The method was evaluated against the commonly used methanol and Chelex methods, regarding polymerase chain reaction detection of Plasmodium falciparum parasites from samples stored for 1-2 years. The sensitivity of detection was dependent on the parasite density and type of filter paper. For 3MM Whatman filter paper, the sensitivity was 100%, 73%, and 93% for the TE, methanol, and Chelex methods, respectively. For the longer stored 903 Schleicher & Schuell filter paper, the sensitivity was 93%, 73%, and 0%, respectively. This rapid, simple, and inexpensive extraction method generated superior results from archived specimens compared with the two standard methods and may represent a useful tool in molecular epidemiologic studies.

Animals↗
PubMed1994

High-throughput rapid yeast DNA extraction. Application to yeast artificial chromosomes as polymerase chain reaction templates.

An automation-assisted method enables DNA extraction of over a thousand yeast colonies in a day by one person. Yeast DNA is extracted essentially quantitatively in three steps: (a) cells are converted into spheroplasts with yeast lytic enzyme, (b) cells are lysed with proteinase K, and (c) DNA is collected by ethanol precipitation. The DNA stored at 4 degrees C is active in polymerase chain reaction experiments for more than a year.

Chromosomes, Artificial, Yeast↗
PubMed1993

Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2001

Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Blotting, Southern↗
PubMed1992

Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions.

We have tested the influence on the polymerase chain reaction (PCR) of a large number of compounds found in food, in media used for selective propagation of food-borne pathogens or in DNA-extraction methods. PCR was found to be sensitive to large volumes of complex food samples containing high amounts of fat and protein, however, an extraction procedure based on treatment with hot NaOH/SDS reduced the effect significantly. Some culture media (Fraser, MLEB, MRB and Rappaport) interfered with the analysis and for most of the media it was possible to assign the inhibitory effect to one or more individual components. Several compounds (detergents, lysozyme, NaOH, alcohols, EDGA, EGTA) used in DNA extraction procedures were found to have some inhibitory effect. The inhibitory effects need to be taken into consideration when designing new tests.

Animals↗
PubMed2000

Fingerprints and DNA: STR typing of DNA extracted from adhesive tape after processing for fingerprints.

An exhibit that is often received for examination in cases of robbery or terrorist activity is adhesive tape. This type of exhibit can often, but not always, be successfully processed for fingerprints. The question arises whether or not it is possible to extract and type DNA after the tape has been sequentially processed for fingerprints. In this work, various donors left fingerprints on the adhesive side of tapes. The tapes were then sequentially processed for fingerprints using an alternate light source, cyanoacrylate fuming, and staining with BY-40 and then crystal violet. DNA was subsequently successfully extracted, amplified and typed for six STR loci.

Adhesives↗
PubMed1988

The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue.

Encouraged by recent reports of extraction of diagnostically useful DNA from formalin-fixed tissues, we devised a study to determine which fixatives are suitable for DNA hybridization studies. Seven lymph node specimens (6 lymphomas and one reactive hyperplasia) were studied. Specimens were divided into portions, each of which was processed separately. Processing protocols followed were: 1) snap freezing; 2) immersion in formalin, ethanol, glutaraldehyde, B5 and methanol acetic acid (MAA) Michel's transport medium or phosphate buffered saline for 2, 24, 48 and 120 hrs.; 3) paraffin embedding following fixation. DNA was obtained by proteinase K digestion followed by phenolchloroform extraction. DNA was cleaved with restriction endonucleases (Eco RI and Bam HI), separated by agarose gel electrophoresis, after which Southern blot hybridization with radio-labelled probes for J-heavy and T-beta genes was performed. Fixation with formalin, glutaraldehyde and B5 resulted in poor yields of DNA. MAA produced degradation of DNA and Michel's medium and PBS gave low quantities and purity of extracted DNA. Ethanol fixation consistently permitted extraction of large amounts of high molecular weight DNA suitable for hybridization studies and compared favourably with the fresh-frozen 'gold standard'. We conclude that ethanol may be the fixative of choice when transport or storage conditions limit the availability of fresh frozen tissue for DNA hybridization studies.

Acetates↗