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At least 217 records · Page 12Linked to original sources
PubMed2002

Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides.

AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.

Animals↗
PubMed1986

Transforming activity of DNA extracted from BKV-transformed hamster cells after passages in culture.

Primary fetal rat fibroblasts (FRF cells) were transfected with DNA extracted from BKV-transformed hamster kidney cells at low passage after transformation (HKBKlp cells) and at high passage after transformation (HKBKhp cells). Two transformed rat cell lines were obtained, FRF-DNA-HKBKlp cells and FRF-DNA-HKBKhp cells, transformed by DNA extracted from HKBKlp cells and HKBKhp cells, respectively. They were characterized by a number of biological properties and by tumorigenicity and metastatic ability in newborn hamsters. The two transformed FRF cell lines exhibited exactly inverted properties in comparison with HKBK cells, in particular FRF-DNA-HKBKlp cells showed a low level of nuclear T antigen and a diffuse appearance of TSTA at the cell surface, whereas FRF-DNA-HKBKhp cells demonstrated a high level of T antigen and the capping of TSTA in the cell membrane. The tumorigencity and the metastatic ability were greater with FRF-DNA-HKBKlp cells, while FRF-DNA-HKBKhp cells appeared to be less tumorigenic and metastatic in newborn hamsters.

Animals↗
PubMed1993

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Aprotinin↗
PubMed1996

Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, GeneReleaser, and the phenol-chloroform method.

The abilities of GeneReleaser and QIAamp to extract the hepatitis B virus (HBV) DNA template from serum for amplification by PCR were evaluated and compared with that of the standard phenol-chloroform method. Differences in the sensitivities of the three methods were revealed by nested PCR of HBV DNA extracted from serially diluted hepatitis B e antigen (HBeAg)-positive (high-titer) serum. Phenol-chloroform was found to be the most sensitive extraction method but was time-consuming and labor intensive, and the many steps required increased the possibility of contamination. In a titration of HBeAg-negative (low-titer) serum, all three methods coupled with nested PCR were capable of detecting low levels of HBV DNA. In the case of QIAamp and GeneReleaser, the extraction was relatively simple and rapid. The higher quantity of serum (200 microliters) used in the QIAamp extraction did not provide higher sensitivity, possibly because of incomplete removal of Taq polymerase inhibitors from the serum or inadequate disruption of the virion. GeneReleaser was more efficient because it gave the same detection limit in low-titer serum as phenol-chloroform even though it utilizes only 5 microliters of serum. However, it did not produce consistent amplifications of HBV DNA, giving false-negative results in 7 of the 50 cases (14%) in one experiment. Use of a larger volume of serum and replicate extractions may overcome this problem. Advantages thus exist in each of the extraction methods, and these should be weighed against the disadvantages when deciding which extraction method is appropriate.

Base Sequence↗
PubMed2005

[Study on detection method of 8-OH-dG in DNA extracted from HepG2 cells in vitro by capillary zone electrophoresis].

OBJECTIVE: To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis. METHODS: HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation. 0 and 15 mmol/L H2O2 were added into two groups of HepG2 cells (5 x 10(7)) respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37 degrees C with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis. RESULTS: The optimized conditions were: uncoated silica capillary (47 cm x 50 microm i.d.), 20 mmol/L borate buffer (pH 9.5), 25 degrees C, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2-untreated DNA increased. CONCLUSION: The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods.

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.

Animals↗
PubMed2004

Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens.

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 of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.

Animals↗
PubMed2005

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis.

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.

Antigens, Bacterial↗
PubMed1999

Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

Centrifugation↗
PubMed2000

Comparison of DNA extraction from blood stain and decomposed muscle in STR polymorphism analysis.

Forensic samples that are often degraded and limited in quality cause DNA typing analysis by conventional methods unsuitable. We performed a single tube-multiplex PCR on 9 STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820) and the X-Y homologous gene amelogenin of DNA extracted from six week postmortem blood stain and decomposed muscle by using QIAGEN QIAamp blood or tissue procedure. An automated genetic analyzer based on fluorescent dye technology was used to detect STR allele patterns. The DNA profile of blood stain sample obtained a complete and unambiguous pattern, whereas, that of muscle DNA extracted from QIAamp tissue and Chelex plus QIAamp blood protocols showed detected STR alleles for 70 per cent and 50 per cent of all tested alleles, respectively. The degraded muscle DNA could not yield amplified products of large size STR alleles; CSF1PO, D13S317 and D7S820. However, the analysis which relied upon the PCR-based STR polymorphism analysis and automated genetic analyzer system offers an ideal strategy for forensic identification.

Autopsy↗
PubMed1974

Transfection of chicken embryo cells with DNA extracted from avian virus-producing neoplastic cells.

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.

Animals↗
PubMed1995

[Application of Plasmodium falciparum DNA extract from dried blood spot of falciparum malaria patient in polymerase chain reaction].

Polymerase chain reaction (PCR) primered with primers 3,4 and 5,6 was performed using DNA extracted from dried blood spot of a falciparum malaria patient from Mengpeng Township, Yunnan using four different methods, including STE-proteinase K-SDS, methanol-proteinase K-SDS, Chelex-100 and Chelex-100-proteinase K to amplify DNA of apical membrane antigen 1 (AMA-1). A 900 bp DNA band could be seen on the ethidium bromide-stained agarose gel electrophoresis of the PCR products when DNA was extracted using all the above-mentioned methods except STE-proteinase K-SDS method. DNA extracted with Chelax-100 was recommended.

Animals↗
PubMed2003

[A study of a brief protocol to extract DNA from hair].

Hair can be a valuable source of DNA for the noninvasive 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. In this study, four different methods for extracting DNA from hairs were compared and the most efficient one was determined. The method for extracting mtDNA from hair shifts was also discussed in the study.

English Abstract↗
PubMed2004

Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction.

Polymerase chain reaction (PCR) has been widely used due to its high specificity, sensitivity, and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. In this study, DNA extraction methods for Mycobacterium avium subsp. paratuberculosis were evaluated including rapid lysis, organic extraction, silica-based and magnetic particle-based (MagaZorb) technologies on bacterial cells, and spiked bovine feces. Efficiency of the extraction was determined by PCR end point titration with primers targeting the insertion sequence, IS900. Results of the end point titrations are identical for bacterial cells and spiked feces. Inhibition was observed in PCR with DNA isolated from spiked feces, and a 1/100 dilution was able to alleviate this problem with DNA extracted by MagaZorb. A 1/1000 dilution was required for the other three methods. MagaZorb proved to be more efficient at removing inhibitory factors and required the least labor and completion time. Further evaluation is required for its utilization in other clinical specimens.

Base Sequence↗
PubMed1990

[Comparative analysis of 2 methods of DNA extraction].

The study of the organization of immunoglobulin and T cell receptor genes in leukaemias and lymphomas depends on our ability to obtain high molecular weight DNA. We have compared two different methods of DNA extraction: one being enzymatic, using the enzyme proteinase K, and the other chemical, using urea as a substance purifying DNA. Since the amount and purity of the DNA obtained are similar with each of the two methods of DNA extraction, both are valid to make such a genetic analysis.

Chemical Precipitation↗
PubMed2006

Meth-DOP-PCR: an assay for the methylation profiling of trace amounts of DNA extracted from bodily fluids.

Cancer cells release their DNA into the patient's bodily fluids and cancer-specific signatures can be recognized in the circulating DNA. The aberrant methylation of CpG-rich regions in gene promoter sequences is an early marker of cell transformation whose specificity and optimal sensitivity can be achieved by assessing the methylation status of multiple genes ('methylation profiling'). Most of the current technologies for methylation analysis rely upon the combination of chemical conversion of the DNA and PCR analysis for the detection of methylated and unmethylated alleles. However, the small amount of circulating DNA, and its fragmentation, dramatically reduces the template DNA molecules making difficult the methylation profiling. To overcome this limitation, we have developed the Meth-DOP-PCR assay, a combination between a modified degenerate oligonucleotide primed PCR (DOP-PCR) and methylation-specific PCR (MSP), for the high-throughput methylation analysis of trace-amount of circulating DNA. We have demonstrated the concordance between Meth-DOP-PCR and MSP and shown the application of this technique for the methylation analysis of DNA extracted from the serum of lung cancer patients. We have estimated that through this procedure it is possible to obtain at least a 25-fold increase of the number of determinations allowing the methylation profiling from less than 1 ml of serum. Thus, Meth-DOP-PCR appears as a simple, cost-effective and efficient technique, for the development of novel methylation-based diagnostic assays.

Blood Chemical Analysis↗
PubMed1978

Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density.

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.

DNA, Superhelical↗