Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA extraction”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources
PubMed1999

A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens.

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.

AIDS-Related Opportunistic Infections↗
PubMed1997

The effect of fixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology.

BACKGROUND: Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is widely used to detect viral genomes, clonal gene rearrangements and oncogene mutations in skin specimens. Fixation with embedding of skin tissue is a procedure that has a profound effect on its molecular arrangement. OBJECTIVE: The aim of this study was to determine the effect of different fixatives on the PCR amplification of DNA. METHODS: We fixed randomly chosen fresh pathologic skin specimens in formalin, ethanol and Histochoice for 24 and 72 h and then embedded the tissue in paraffin. DNA was extracted from the paraffin-embedded tissues and used as template for amplification, producing 530- and 760-bp fragments of the phosphoglycerokinase gene. RESULTS: Our results indicate that PCR can be performed with excellent results on ethanol- and Histochoice-fixed, paraffin-embedded skin tissue with a rate of success comparable to that using fresh tissues; formalin-fixed tissue gave slightly less satisfactory results. CONCLUSION: This investigation corroborates previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that DNA extracted from tissue fixed with Histochoice is suitable for PCR gene amplification.

Base Sequence↗
PubMed1984

Restriction enzyme analysis of human cytomegalovirus using DNA extracted from infected cells.

Cytomegalovirus (CMV) isolates were investigated by restriction enzyme analysis. A method for extraction of cellular CMV DNA is described which uses CMV DNA released into the Hirt supernatant after precipitation of cellular DNA. CMV DNA is further purified by ultracentrifugation in neutral sucrose gradient. Fifteen strains from unrelated patients all had unique cleavage patterns with three restriction enzymes, although many of the fragments comigrated. Strains from related patients mostly showed identical patterns. The CMV genome seems to be stable since the pattern did not change in five isolates obtained from the same patient during a 5-year period.

Base Sequence↗
PubMed2000

Rapid extraction of DNA from archival clinical specimens: our experiences.

The analysis of DNA extracted from archival clinical specimens using polymerase chain reaction represents the basis of a variety of research and diagnostic protocols in medicine. However, the selection of optimal DNA extraction method is critical if such an analysis is to be successful. Recently, we have evaluated a number of rapid DNA extraction protocols in order to find the most suitable method for routine processing of the most common archival materials in pathological and cytological laboratories: paraffin-embedded tissues and Papanicolaou- or Giemsa-stained smears. Our results demonstrate that rapid DNA extraction methods have comparable DNA extraction efficiencies with standard DNA isolation protocols on archival clinical specimens with the exception of Giemsa-stained smears.

Archives↗
PubMed2003

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction.

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Bacteria↗
PubMed1990

[Sex identification by analysis of DNA extracted from hard tissues].

Sex identification of unidentified two bleached skeletons by the method of DNA analysis in addition to the morphological examination was performed. Bones and teeth were taken out for the DNA analysis, and cracked to compounds by liquid nitrogen. DNA was extracted by the standard procedure. We used two methods for sex identification by DNA analysis; one is Southern blot hybridization with Y-chromosome specific probe (PHY10; 3.4kb), and the other is PCR (polymerase chain reaction) amplifying with sex chromosome specific fragments (alphoid satellite family). These two methods enable us to identify the sex of the two cases presented here more correctly with sufficient evidence than depending only on morphological examination. With some technical difficulties, it is possible to put DNA analysis including Southern blot hybridization or PCR into practical determination of sexing of hard tissues such as a piece of bone or a tooth, and old samples. Therefore, they seem to be effective methods applicable to forensic science.

Adolescent↗
PubMed2000

New protocol for DNA extraction of stool.

Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.

Base Sequence↗
PubMed1990

[DNA extraction from body fluids of cadavers].

DNA of 1 ml Ascites, bile, liquor, pleural-exsudate, pericardial-fluid, synovial-fluid, urine and vitreous humor of sixteen bodies of either sex was isolated. Applying dot-blot-hybridisation black spots were obtained by using total human DNA as DNA-probe (A), if the origin is human. Our results showed, that body-fluids of dead persons contain human DNA, mainly. After rehybridisation with a sex-specific DNA-probe (Y) (pJA 1143) a determination of sex can be performed.

Body Fluids↗
PubMed2006

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

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

DNA, Plant↗
PubMed1989

DNA extraction from bloodstains in respect to age and stained substrate.

The amount and the quality of DNA that could be extracted from bloodstains equivalent to 200 microliters blood were examined after 3, 6, 9, 12, 15, 18 and 21 days of storage under dry or humid conditions at room temperature. DNA was also extracted from two days old 200 microliters bloodstains on different stain carriers. The amount of DNA that could be extracted showed a dependency on all the parameters that were examined. On carriers with a rough surface structure, where the blood can soak in, the resolving of leukocytes was impaired and the DNA recovery rate was low, e.g. carpeting and suede. The DNA yield was higher for substrates with a smooth surface, e.g. paper, glass and smooth leather. The aging experiments revealed that for the stains stored under dry conditions, there was no decrease in the DNA yield for stains on glass, while the amount of DNA that could be extracted became less for older stains on cotton. Under humid conditions the DNA yield was high and did not decrease for both glass and cotton fabric. The quality of the DNA was not impaired by aging or storage conditions, but for several stain carriers the DNA was contaminated by chemical substances which inhibited restriction enzyme digestion and successful DNA-typing.

Aging↗
PubMed1995

Use of a rapid and simple method to extract proviral DNA in the identification of HIV-1 by PCR.

DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV-1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity.

Adult↗
PubMed2002

Accumulation of somatic mutation in mitochondrial DNA extracted from peripheral blood cells in diabetic patients.

AIMS/HYPOTHESIS: A point mutation of mitochondrial DNA at nucleotide number 3243 A to G is responsible for both the major genetic aetiologies of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and mitochondrial diabetes. Otherwise, this mutation is also reported to occur as an acquired somatic mutation, possibly due to oxidative stress. Since diabetes can cause severe oxidative stress, we hypothesize that the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA can be accelerated by diabetes. METHODS: DNA was extracted from blood samples of 290 non-diabetic healthy subjects (age 20-60) including 98 newborn infants and from 383 patients with Type II (non-insulin-dependent) diabetes mellitus (age 18-80). The extent of somatic 3243 A to G mutation to total mitochondrial DNA was detected by real-time PCR using the TaqMan Probe. RESULTS: Whereas the level of the 3243 A to G mutation was negligible in the newborn group, it was increased in healthy subjects who were 20 to 29 and 41 to 60 years of age, suggesting that this mutation was somatic. In the diabetic patients the mutation rate increased along with age and the duration of diabetes. In the middle-aged group (age 41-60), the 3243 A to G mutation accumulates fourfold higher in the diabetic patients than the healthy subjects. Moreover, multiple regression analysis showed that the most critical factor associated with this mutation in diabetic patients was the duration of diabetes. CONCLUSION/INTERPRETATION: Diabetes accelerates the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA, which can accelerate the ageing process. This somatic mutation could possibly be a new marker for estimating the duration of diabetes.

Adenine↗
PubMed1993

SV40 DNA extracted from persistently infected Vero cells using miniprep columns for plasmids.

A recombinant pAT153 plasmid carrying the whole simian virus 40 (SV40) genome in the form of two consecutive Pst I restriction fragments was rapidly isolated using a commercially available minipreps DNA purification system. This rapid and simple method was used to extract SV40 DNA from infected Vero monkey cells. Viral DNA replication in persistently infected SVP-1 monkey cells was also conveniently followed over a period of 8 d by agarose gel electrophoresis and molecular hybridization using minipreps and the recombinant plasmid (pLCB104) as a probe. Unsatisfactory results were obtained, however, when herpes simplex virus DNA was tentatively extracted from infected Vero cells with the above method. Only covalently closed circular DNA molecules, with two strands unable to separate fully under denaturing conditions, were apparently retained after rapid neutralization by the silicone-based minipreps DNA purification resin.

Animals↗
PubMed1990

A new method for extracting DNA or RNA for polymerase chain reaction.

The use of glass powder suspension for the extraction of RNA or DNA was studied to simplify the procedures of polymerase chain reaction (PCR). Using this procedure, proviral DNA of human T-lymphotropic virus type-1 (HTLV-1) in the blood of an asymptomatic virus carrier and viral RNA of human immunodeficiency virus (HIV) in the blood of an AIDS patient were easily detected by PCR employing glass powder. The use of glass powder is a simple and highly efficient procedure for the extraction of DNA or RNA, and can be applied for routine PCR.

Acquired Immunodeficiency Syndrome↗
PubMed1997

DNA extraction from liquid blood using QIAamp.

The implementation of convicted felon DNA databases by increasing numbers of forensic science laboratories has engendered the need for a quick, efficient, and cost-effective method for the isolation of DNA from liquid blood samples. Because of the large numbers of samples involved, the ideal method would combine high throughput capability with maximal yield, high quality, and minimal time. We have found that the QIAGEN QIAamp Blood Kit/Tissue Kit satisfy all of these requirements. This simple, low cost spin column procedure yields purified DNA of approximately 20-30 kb that can be used directly in PCR or other enzymatic reactions without further purification. We compared the QIAamp isolation procedure to the standard SDS-Proteinase K/organic extraction/microcon purification procedure currently used by many forensic laboratories. The QIAamp procedure consistently gave a two- to four-fold increased yield relative to the organic extraction procedure. The DNA obtained was of high molecular weight, exhibited little degradation, and was suitable for RFLP and PCR analyses. We have found QIAGEN's QIAamp DNA isolation procedure to be ideally suited for preparation of samples for DNA databasing.

DNA↗
PubMed2004

A rapid DNA extraction method for PCR identification of fungal indoor air contaminants.

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) applications. The methodology described is both rapid and cost effective for use with multiple fungal organisms.

Air Microbiology↗
PubMed1969

Human diploid cell transformation by DNA extracted from the tumor virus SV40.

The DNA isolated from simian virus 40 (SV40) can transform human fibroblast cells in tissue culture. Clonal lines of DNA-transformed human cells have been obtained that have properties characteristic of cells transformed by whole virus. They all contain SV40 T-antigen, and infectious virus can be recovered by cocultivation. This is the first demonstration of a permanent genetic alteration produced in human cells by purified DNA.

Animals↗