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At least 271 records · Page 15Linked to original sources
PubMed2005

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots.

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.

DNA, Fungal↗
PubMed1978

Dextran sulfates as a contaminant of DNA extracted from concentrated viruses and as an inhibitor of DNA polymerases.

Dextran sulfate is commonly used with polyethylene glycol to concentrate viruses before extraction of their DNA. However, dextran slulfate then easily contaminated such DNA and acted as a potent inhibitor of DNA polymerases from Bacillus subtilis (III), phage PBS2, and phage T4. Dextran sulfate only weakly inhibited Micrococcus luteus and Escherichia coli DNA polymerase I preparations.

Bacteriophages↗
PubMedDate not supplied

[Method for rapid DNA extraction from bacterial communities of different soils].

A method for indirect DNA extraction from various soils significantly differing in their physicochemical properties has been developed. The proposed method is based on cell desorption from soil particles using a Tris-EDTA (TE) buffer supplemented with polyvinylpolypyrrolydone (PVPP) and sodium dodecylsulfate (SDS). Subsequent cell lysis and purification of DNA preparations methods based on alkaline lysis followed by chromatography on ion-exchange resins were described by us earlier. The purity of the DNA preparations obtained did not depend on the type of soil. It was shown that the DNA preparations can be used for the amplification of rather large fragments, e.g., sequences spanning the complete 16S rRNA gene.

Bacteria↗
PubMed2002

DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound.

Magnetic particles produced by magnetic bacteria have been used to carry out magnetic separation of DNA. Separation was achieved using magnetite coated with 3-aminopropyltriethoxysilane, N-(trimethoxy-silylpropyl) isothiouronium chloride or 3-[2-(2-aminoethyl)-ethylamino]-propyltrimethoxysilane (AEEA). The DNA binding efficiency increased with the number of amino groups present on the silane compounds and was 14 fold higher than with untreated magnetite. Addition of AEEA to aqueous solutions containing coated magnetite increased efficiency due to co-condensation of DNA. From 10(8) Escherichia coli cells, 7.1 microg of DNA was recovered using 100 microg of magnetite. E. coli DNA extracted with modified bacterial magnetite was suitable for restriction enzyme digestion and polymerase chain reaction (PCR). Ten replicate samples of E. coli cells were extracted using an automated magnetic robot and 9.5 microg of DNA was extracted using 100 microg of modified bacterial magnetite and possessed a 1.94 absorbance ratio (260:280 nm).

Amines↗
PubMedDate not supplied

[Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

DNA↗
PubMed2003

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales.

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.

Animals↗
PubMedDate not supplied

Southern blot analysis of DNA extracted from paraffin wax-embedded tissue.

Genetic analysis of multi-generation families using RFLP is often incomplete because fresh tissue is not available for the isolation of DNA. However, in many instances archival material exists, usually formalin-fixed in paraffin blocks. Procedures have been described for extracting DNA from such specimens. Many parameters affect the quality of the DNA eventually extracted and this in turn determines whether the DNA can be successfully digested with restriction enzymes and probed (Dubeau et al., 1986; Warford et al., 1988). In essence, the procedures followed at the time of fixation largely determine whether archival material will yield useful results. We have re-examined the effect of varying the fixation time but, more importantly, we have assessed the validity of RFLP results obtained from fixed tissue.

Blotting, Southern↗
PubMed2005

On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field.

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.

DNA↗
PubMed1992

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

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

Adolescent↗
PubMed2004

Rapid determination of the Delta32 deletion in the human CC-chemokine receptor 5 (CCR5) gene without DNA extraction by lightcycler real-time polymerase chain reaction.

CCR5 is a major coreceptor for cellular entry of macrophage-tropic isolates of the human immunodeficiency virus (HIV). A 32-base pair deletion of the CCR5 gene (CCR5Delta32) protects against HIV infection because the frame shift leads to a truncated protein not expressed on the cell surface. CCR5Delta32 also delays progression in heterozygous HIV-infected patients and improves responses to antiretroviral therapy. Available methods for CCR5 genotyping, however, are cost expensive and/or time consuming. To improve CCR5 genotyping we studied four primer sets flanking the CCR5Delta32 deletion site using real-time polymerase chain reaction (PCR) on a LightCycler device. Primers amplified fragments of different length depending on the presence or absence of the Delta32 mutation. Next, melting curves of the amplified fragments were analyzed using SYBR green, a conventional double-strand DNA dye. To circumvent initial DNA extraction, we also studied serially diluted "interphase" leukocytes as PCR templates after centrifugation of EDTA blood. Finally, the validity of the new method was checked by analyzing 100 blood samples with known CCR5 genotypes. Amplicons of 82 bp:50 bp and 97 bp:65 bp fragment ratios could easily be discriminated due to the differences in their melting temperatures (3 degrees C and 2 degrees C, respectively). Furthermore, CCR5 genotyping was possible without initial DNA extraction and yielded optimal results at 1:400 to 1:600 dilution of the "interphase" leukocytes. Results of the new LightCycler PCR protocol were identical to conventional CCR5 genotyping but required considerably less time and expenditures. We have established a new real-time PCR protocol, which enables fast, cost-saving, and reliable CCR5 genotyping.

DNA Primers↗
PubMed1997

A rapid typing system at three STR loci from bloodstains using a simple DNA extraction kit and capillary electrophoresis.

A rapid typing method for STR analysis from bloodstains was devised. DNA from three-year-old bloodstains of six individuals was extracted with a cationic detergent in a newly-released kit. Amounts of DNA extracted by the kit were 16.2 +/- 1.8 ng from 10 microliters of fresh blood samples. DNA was also extracted from bloodstains of three months and three years left at room temperature, and amounts of extracted DNA were estimated at 2.2 +/- 1.0 ng and 0.1 +/- 0.1 ng, respectively. The PCR mixture supplied with the kit, plus three sets of fluorescently labeled primers, were directly added to the tube used for DNA extraction. The amplified products were then analyzed by capillary electrophoresis, and the obtained data were automatically sized and typed using computer software. Three genotypes of all bloodstains except one sample at a single locus were determined correctly as those of freshly collected blood from the same individuals although some stains contained no more than 0.1 ng. Using this typing system, we were able to type three STR loci, HUMTH01, HUMF13A1 and HUMFES/FPS, from a single bloodstain within five hours. This typing is particularly useful for forensic practice.

Blood Stains↗
PubMed1998

A spin cartridge system for DNA extraction from paraffin wax embedded tissues.

A simple and efficient method of DNA extraction from paraffin wax embedded tissues using a spin cartridge system is described. Such DNAs were shown to be suitable for amplification by the polymerase chain reaction, which targeted two human papillomavirus genes and one globin fragment giving rise to products of 450, 150, and 110 base pairs, respectively. Different human tissues, stored for up to 20 years, were successfully amplified, demonstrating the usefulness of this very simple procedure for retrospective studies.

Centrifugation↗
PubMed2006

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

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.

Animals↗
PubMed1996

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

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

Animals↗
PubMed2005

A simplified method for mitochondrial DNA extraction from head hair shafts.

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 treatments. A 203 bp mtDNA product could be amplified from 90% of samples extracted by alkaline digestion and 73% of hairs extracted by glass grinding. DNA obtained from alkaline digested hair generated equal or greater amplification success for virtually all criteria examined, and mtDNA sequences matched buccal control sequences in all cases. The two methods were similar in DNA yield (amplification success at template dilution) and quality of DNA obtained (amplicon length). Alkaline digestion of hair shafts required 6-7 h to complete, compared to 22-24 h for glass grinding, and proved a less laborious yet equally robust method for mtDNA extraction.

Adult↗
PubMed2004

Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation. OBJECTIVES: The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR). STUDY DESIGN: First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14). RESULTS: A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3. CONCLUSION: This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).

AIDS-Related Opportunistic Infections↗
PubMed1989

Use of silica gel polymer for DNA extraction with organic solvents.

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.

Chemistry Techniques, Analytical↗
PubMed1993

A rapid DNA extraction method from culture and clinical samples. Suitable for the detection of human cytomegalovirus by the polymerase chain reaction.

We propose an one-step DNA extraction method suitable for the polymerase chain reaction. This procedure utilizes Chelex 100, a chelating in exchange resin. This technique was compared with a traditional technique (proteinase K lysis, phenol-chloroform extraction and ethanol precipitation) for isolation of human cytomegalovirus DNA from clinical samples. The procedure using Chelex 100 appeared to be a simple and fast extraction method for human cytomegalovirus DNA.

Bronchoalveolar Lavage Fluid↗