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At least 199 records · Page 11Linked to original sources
PubMed2001

DNA extraction from soils: old bias for new microbial diversity analysis methods.

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

Bacteria↗
PubMed1972

In vitro repair of x-irradiated DNA extracted from Bacillus subtilis deficient in polymerase I.

DNA extracted from x-irradiated cells of a mutant of Bacillus subtilis deficient in DNA polymerase I has greatly reduced biological activity. This DNA, which has many single-strand nicks, can be repaired in vitro with purified DNA polymerase I and DNA ligase or with lysates of wild-type B. subtilis cells, which restore biological activity and increase the single-strand molecular weight. Lysates of polymerase I-deficient B. subtilis cells cannot repair such irradiated DNA until purified DNA polymerase I or lysate from wild-type cells is added to the deficient lysate.

Bacillus subtilis↗
PubMed2004

Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method.

Animals↗
PubMed1995

Reliability of differential PCR for the detection of EGFR and MDM2 gene amplification in DNA extracted from FFPE glioma tissue.

A series of 43 human gliomas, consisting of 30 glioblastomas, 7 anaplastic astrocytomas, 3 low grade astrocytomas, 2 ependymomas, and 1 oligodendroglioma, was studied for amplification of the epidermal growth factor receptor (EGFR) and mouse double minute 2 (MDM2) genes. DNA extracted from formalin-fixed, paraffin-embedded tissue sections was analyzed by differential PCR and the results were compared with slot blot examination of DNA extracted from frozen tissue from the same neoplasms. Twelve glioblastomas (40%) showed amplification of the EGFR gene, and overexpression of EGFR was evident in each of these tumors as indicated by the immunoperoxidase technique. Two of the tumors with EGFR gene amplification also revealed amplification of the MDM2 gene, while one additional glioblastoma revealed MDM2 amplification only. A 100% concordance in the detection of amplification was observed between differential PCR and slot blot analysis; consequently, these results indicate that differential PCR using DNA extracted from archival tissue sections is a reliable method of demonstrating gene amplifications in glial tumors.

Adult↗
PubMed2003

A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus.

In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.

Animals↗
PubMed2001

Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation.

All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 microg g(-1) dry weight soil versus 38-93 microg g(-1) obtained by in situ lysis methods). However, with the exception of the gamma-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.

Bacteria↗
PubMed2004

Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in Rwanda.

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the pol region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.

Acquired Immunodeficiency Syndrome↗
PubMed2005

The "spanning protocol": a new DNA extraction method for efficient single-cell genetic diagnosis.

PURPOSE: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." METHODS: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. RESULTS: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. CONCLUSION: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Blastomeres↗
PubMed1999

Rapid DNA extraction for molecular epidemiological studies of malaria.

DNA isolation from blood samples collected in molecular epidemiological studies is crucial for the quality and reproducibility of data. Blood samples from two malaria endemic sites have been prepared by four different DNA isolation methods with subsequent PCR amplification of the msp2 locus of Plasmodium falciparum. We tested a rapid boiling method; the guanadine isothiocyanate DNA extraction; QIAmp blood kit; and the ISOCODE STIX PCR template preparation dipstick, and analysed the numbers of concurrent infections/sample. The rapid boiling method and the ISOCODE STIX provided overall the best sensitivity combined with ease of handling. The possibility to store and ship the ISOCODE STIX at ambient temperature adds further advantage to this method.

Animals↗
PubMed1994

Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR.

AIMS: To evaluate the ability of four rapid DNA extraction methods to provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues. METHODS: Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. Different exons of the p53 gene, human papillomavirus type 16 (HPV 16) sequence, and immunoglobulin heavy chain (IgH) gene rearrangement were amplified from the extracts. RESULTS: The Chelex boiling, proteinase K digestion, and proteinase K digestion-Chelex boiling methods produced DNA suitable for amplification in all of the 45 samples. Boiling in water yielded insufficient template for the PCR in three of the 45 cases (7%), and in six of 42 positive cases (14%) much fainter bands were observed, mostly when the processed material was either biopsy specimen sized or a B cell lymphoma sample. Fragments of the p53 gene were successfully amplified up to 408 base pairs in water boiled extracts, up to 647 in Chelex boiled preparates, and up to 984 in proteinase K digested and proteinase K digested-Chelex boiled samples, although with decreased sensitivity in the last case. All of the templates were reusable after 3 months of storage at -20 degrees C. CONCLUSIONS: Chelex boiling, proteinase K digestion, and proteinase K digestion followed by Chelex boiling produce suitable templates for the PCR from a large variety of paraffin wax embedded tissues. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material.

Base Sequence↗
PubMed2006

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

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 16th to the early 19th centuries. In parallel extractions including or excluding a washing step were performed at least two times for each sample in order to evaluate the influence on the quantity of nuclear DNA yielded and on the typing efficiency. Twelve ancient individuals were analyzed through our approach and full and reliable profiles were obtained for four of them. These profiles were validated by comparison with those obtained from bone and teeth DNA extracted from the same ancient specimens. The present study demonstrates that the washing step cannot be considered as deleterious for DNA retrieval since the same results were obtained by the two approaches. This finding challenges the hypothesis that recoverable nuclear DNA is only found on the outer surface of hair shafts and provides evidence that nuclear DNA can be successfully extracted from ancient hair shafts. The method described here constitutes a promising way for non-invasive investigations in ancient DNA analysis for precious or historical samples as well as forensic casework analyses.

Bone and Bones↗
PubMed2005

DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

Base Sequence↗
PubMed2006

A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: methods for DNA extraction and denaturing gradient gel electrophoresis analysis.

The diversity of the phytobenthonic community present in six acidophilic microbial mats from Río Tinto (Iberian Pyritic Belt, SW Spain) was analysed by optical microscopy and two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 18S rDNA cloned gene fragments. Sixteen DNA isolation protocols as well as two commercial DNA extraction kits were tested and their efficiency compared. Purified DNA extracts were amplified by PCR using universal eukaryotic primers and the PCR products analysed by DGGE. Bead-mill homogenization was found to be superior to the other cell lysis methodologies assayed (sonication or freeze-thawing cycles) as it allowed efficiencies of cell disruption of over 95%. The methods combining bead-mill homogenization in the presence of SDS, treatment with chemical extractants (hexadecylmethylammonium bromide or guanidine isothiocyanate) and phenol extraction resulted in DNA preparations that amplified the same number of bands when analysed by DGGE as the two commercial kits assayed. The phylogenetic affiliations of the DGGE bands were determined by a BLAST search, and nine different species related to the Chlorophyta, Ciliophora, Kinetoplastida, Ascomycota, Streptophyta and Colcochaetales taxonomical groups were identified. Similar levels of diversity were found using cloning procedures. Although not all the species observed under the microscope were detected using molecular techniques, e.g. euglenas, heliozoan, or amoebae, DGGE fingerprints showed rather well the level of diversity present in the samples analysed, with limitations similar to cloning techniques.

Animals↗
PubMedDate not supplied

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

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.

Algorithms↗
PubMed2004

[Cryptococcus spp. DNA extraction from environmental samples].

The genus Cryptococcus encompasses 38 species, but only 3 are associated with disease in humans and animals, Cryptococcus laurentii, Cryptococcus albidus and Cryptococcus neoformans. The last one is the most frequently reported. The disease is acquired by the inhalation of infectious propagules present in the environment. The habitat has been established using extraction techniques with buffer supplemented with antibiotics and plating in selective media. The aim of this work was to evaluate several DNA extraction techniques for Cryptocococus spp. from environmental samples. The control isolates were C. neoformans, C. albidus, C. laurentii and Paracoccidiodes brasiliensis. We also used vermiculita and soil samples contaminated with different yeast concentrations (10 to 10(6) cells/g) and samples naturally contaminated with C. neoformans. DNA was extracted with physical and chemical methods and with a commercial kit, and the DNA was purified with agarose blocks and silica columns. For the PCR amplification we used the CN4-CN5 primers, which are specific for C. neoformans. Only the commercial kit allowed DNA extraction and amplification from contaminated soil samples up to a concentration of 10 cells/g and from one sample naturally colonized. With this work we extracted and amplified DNA from Cryptococcus spp. from environmental samples with appropriate PCR specificity, it will be a tool to establish the ecological areas of C. neoformans in our country.

Case-Control Studies↗
PubMed1997

DNA extraction by zinc.

A fast, very simple and efficient method of DNA extraction is described which takes advantage of DNA sedimentation induced by millimolar concentrations of ZnCl2. The zinc-induced sedimentation is furthermore strongly promoted by submillimolar phosphate anion concentrations. Within <30 min, the method recovers >90% of DNA irrespective of whether a plasmid DNA or short oligonucleotides are the extracted material. The method works with plasmid DNA and oligonucleotide concentrations as low as 100 ng/ml and 10 microg/ml, respectively, without using any expensive facilities or toxic chemicals.

Buffers↗
PubMed1997

Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR.

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.

AIDS-Related Opportunistic Infections↗
PubMed2000

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones.

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500-1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.

Adolescent↗