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PubMed1990

Extraction of nucleic acids from agarose gel--a quantitative and qualitative comparison of four different methods.

Agarose gel electrophoresis is commonly used to separate different species of nucleic acids. We compare four different methods of extraction which are commonly used. These methods include buffer extraction, electroelution, glass bead extraction and extraction of DNA from low-melting agarose. The results show that DNA extracted by these four methods is comparable in their ligability to the PMT 21 vectors and the plasmids with insert can be used for subsequent transfections of competent bacteria. There is a higher yield for buffer extraction and electroelution when compared with glass bead extraction and low melting agarose (p less than 0.05). To conclude, the four commonly used methods for DNA isolation are comparable qualitatively. But the simplest method, namely buffer extraction, has the highest yield.

Bacteriophages↗
PubMed1998

[Studies on cloned genomic differential DNA fragments extracted from esophageal carcinoma and non-cancerous esophageal tissues in Linxian County].

OBJECTIVE: To isolate the genes involved in carcinogenesis of esophageal cancer and to provide a molecular marker for screening and early detection of it in susceptible individuals. METHODS: DNA extracted from 12 matched pairs of esophageal cancer and non-cancerous tissues in patients of Linxian County, Henan Province, a high incidence area for esophageal cancer, and differential DNA fragments were isolated with arbitrary primer polymerase chain reaction (AP-PCR), cloned and sequenced. Restriction endonuclease Dra I and Hind III were used to digest genomic DNA in the pair-matched cancerous and non-cancerous esophageal tissues for Southern blot analysis. RESULTS: There was a randomly amplified differential DNA fragment of about 1.0 kb in cancerous tissues in three of the twelve cases with esophageal cancer, but no such fragments could be found in corresponding non-cancerous tissues. Sequence analysis showed that no homologous DNA in GeneBank database as same as 11T differential DNA fragments. Southern blot analysis showed that there was significant difference between pair-matched cancerous and non-cancerous tissues. CONCLUSION: 11T differential DNA fragment appeared during carcinogenesis of the esophagus, and further investigations were needed to study if it could be regarded as a new kind of oncogene or a biomarker of it.

Base Sequence↗
PubMed2002

A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

Actinomycetales↗
PubMed2004

Optimisation and validation of methods to assess single nucleotide polymorphisms (SNPs) in archival histological material.

BACKGROUND AND PURPOSE: An increasing amount of evidence indicates that single nucleotide polymorphisms (SNPs) may affect a variety of oncology related phenotypes. Occasionally, it is convenient to base studies addressing genotype-phenotype relationships on historical patient cohorts, from which only archival specimens are available. This study was conducted to validate protocols optimised for assessment of SNPs based on paraffin embedded, formalin fixed tissue samples. PATIENTS AND METHODS: In 137 breast cancer patients, three TGFB1 SNPs were assessed based on archival histological specimens. In 37 of these patients, the SNPs were also assessed using cultured fibroblasts and the assays were validated by direct comparison of the results. From the remaining 100 patients, only archival material was available. In these patients, the existence of a genetic linkage pattern between the assessed TGFB1 SNPs was used to provide an indirect validation of the genotyping results. Furthermore, two different methods for DNA extraction were compared (semi-automatic DNA extraction using the ABI Prism 6100 Nucleic Acid PrepStation versus Proteinase K digestion for 5 days followed by boiling and DNA precipitation). RESULTS: Assessment of SNPs based on archival histological material is encumbered by a number of obstacles and pitfalls. However, these can be widely overcome by careful optimisation of the methods used for sample selection, DNA extraction and PCR. Within 130 samples that fulfil the criteria for analysis a highly reliable SNP assessment was observed. The study demonstrated that different 'down-stream applications' ('single nucleotide primer extension' or 'TaqMan-based' real-time PCR) could be used as genotyping procedure. CONCLUSIONS: Reliable assessment of SNPs in formalin-fixed paraffin-embedded specimens is possible but a number of precautions should be carefully taken.

Biological Specimen Banks↗
PubMed1998

A novel method of extracting plasmid DNA from Helicobacter species.

BACKGROUND: Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. MATERIALS AND METHODS: Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. RESULTS: High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. CONCLUSION: This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.

Amino Acid Sequence↗
PubMed2005

Comparison of LightCycler PCR and culture for detection of group B streptococci from vaginal swabs.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.

Bacterial Typing Techniques↗
PubMed1976

DNA synthesis in vitro dependent upon phiX174 replicative form I DNA.

Extracts of Escherichia coli strains infected with bacteriophage phiX174 catalyze DNA synthesis dependent on double-stranded, circular phiX174 replicative form I (phiX RFI) by a semiconservative process. The reaction required Mg++, ATP, all four dNTP, and exogenous phiX RFI DNA as template and yielded phiX RFI and phiX RFII. The reaction was inhibited by nalidixic acid and novobiocin but not by rifampicin. DNA synthesis required the phiX174 gene A product and E. coli gene products dnaB, dnaC(D), dnaG, and rep.

Bacterial Proteins↗
PubMed1976

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.

Animals↗
PubMed2000

Use of archival tissue in epidemiologic studies: collection procedures and assessment of potential sources of bias.

Collection and analyses of archival tumor tissue as a means to increase our understanding of disease pathways is becoming an important avenue of epidemiologic research. In this paper, we present methods of collection and processing of archival tissue and assess the population characteristics of those for whom we were able to and unable to obtain tumor DNA. Cases of colon cancer diagnosed between September, 1991 and October, 1994 living in Utah, Northern California, or the Twin Cities Metropolitan area of Minnesota were targeted for this study. Of the 2477 people for whom we had permission to obtain tumor blocks, we were able to collect blocks and extract DNA for 2117 (85.5%). There were no differences in age, tumor site, or diet and lifestyle characteristics between those with and without DNA extracted. However, we were less likely to be able to extract DNA if the case was diagnosed at a more advanced disease stage or at the earliest disease. Potential bias from exclusion of those with the most advanced disease stage is discussed.

Adult↗
PubMed1996

The difficulty of detecting HTLV-1 proviral sequences in patients with mycosis fungoides.

Although most patients with cutaneous T cell lymphomas, including mycosis fungoides (MF) and its leukemic variant, the Sézary syndrome, are seronegative for antibodies to the human T cell lymphotropic viruses (HTLV-I/II), it has recently been shown that > 95% of such patients harbor proviral DNA sequences related to the region of the HTLV genome that encodes the transregulatory/transforming gene, tax. However, the demonstration of HTLV sequences, even after amplification by polymerase chain reaction (PCR), has not been universally successful, and some investigators continue to question this observation. In an effort to resolve this controversy, we have compared published methodologies that have been less successful with techniques currently used in this laboratory. Major differences were found in (a) the nature of the cells used [freshly isolated versus cultured peripheral blood mononuclear cells (PBMC)] and (b) the methods used to prepare samples for PCR (whole cell lysates versus DNA extracts). PBMC from 10 different MF patients and the healthy daughter of 1 of the patients were subjected to comparative analyses. While all of the PBMC lysates were positive, the DNA extract from only one of these individuals revealed HTLV tax sequences. Studies were also conducted comparing cell lysates and DNA extracts of cultured cells derived from tax sequence-positive PBMC from seven different MF patients. The cells from four of the seven were shown to have retained tax sequences after varying times in culture, when whole-cell lysates were used as targets for PCR amplification and Southern analysis, whereas none of the DNA extracts were positive. It appears that the use of whole-cell lysates instead of DNA extracts and the use of fresh instead of cultured cells greatly enhance the ability to detect HTLV-1 tax sequences in specimens from MF patients.

Blotting, Southern↗
PubMed2002

The difference in the stimulation by putrescine of DNA synthesis using DNA polymerase extracts of normal rat liver or of tumour tissue or host liver from tumour-bearing rats.

Putrescine biosynthesis is elevated before DNA replication, and a stimulation of DNA synthesis by 20 mM putrescine has been found using an in vitro DNA synthesizing system. Furthermore, this stimulation of DNA synthesis by putrescine involves a particular factor (factor PA). This factor PA stimulates DNA polymerases alpha, beta, and gamma, and is present in nuclei and mitochondria but not in cytoplasm. Factor PA loses about 80% of its activity by heating at 45 degrees C for 15 min or by hydrolysis with 100 mg ml(-1) Enzygel trypsin. These properties indicate that factor PA is a protein. Its size is estimated to be about 2.1 S. DNA synthesis in nuclear and mitochondrial DNA polymerase extracts from tumour tissues and host livers of tumour-bearing rats are not stimulated by 20 mM putrescine. However, the addition of excess factor PA to DNA synthesizing systems using DNA polymerase extracts from proliferative tissues again results in a stimulation of DNA synthesis by exogenous putrescine. These findings indicate that the stimulatory effect of DNA synthesis in vitro by exogenous putrescine is controlled by the ratio between factor PA and endogenously synthesized putrescine in proliferative tissues or that sent by the bloodstream from proliferative tissues. These results suggest that a non-stimulatory effect of putrescine on DNA synthesis may be diagnostic in tumour-bearing patients.

Animals↗
PubMedDate not supplied

Improved assessment of isolated islet tissue volume using digital image analysis.

Accurate and consistent measurement of tissue volume is critical to performing many types of islet research; however, conventional visual determination of isolated islet yields through a microscope is heavily operator dependent. An improved method of islet volume determination using digital image analysis (DIA) was developed to remove operator bias and automate the islet counting process. A series of 140 porcine islet isolations were used to evaluate the DIA method in three separate stages. In Stage 1 (n = 29 isolations), the conventional and DIA methods were correlated with two other independent islet quantitation methods: insulin extraction, and DNA extraction. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally determined volumes. In Stages 2 and 3 (n = 54 and 57 isolations, respectively), it was shown that an increase in the number of fields analyzed by DIA did not significantly improve the quality of the correlations. Inclusion of very small tissue (<50 microm in diameter), which is ignored in the conventional protocol affected yields by less than 10% and did not significantly improve the correlation with insulin or DNA content. Quantitation of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of transplanted tissue.

Animals↗
PubMed2001

Extraction of DNA from oral cytological samples by scraping and smear method suitable for restriction site mutation analysis: a pilot study.

The restriction site mutation assay (RSM) can be used to measure base changes which occur in the DNA coding for bacterial restriction enzymes. The aim of this study was to investigate whether DNA, of sufficient quantity and quality for analysis by RSM, could be extracted from cells collected from precancerous lesions using a cytological smear technique. Six smears were collected from each of five lesions of leukoplakia displaying a variety of clinical appearances. Three methods for the extraction of DNA were compared. The commercial extraction method was shown to be most convenient and reproducible, routinely providing 1-5 microg of DNA per sample. Cell populations collected by a cytological smear technique can provide DNA of sufficient quantity and quality for analysis of RSM.

DNA↗
PubMed1993

Identification and quantitation of apoptotic cells following anti-CD3 activation of murine G0 T cells.

Multiparameter flow cytometry and cell sorting were used to examine the process of apoptosis after activation of murine resting T cells with immobilized anti-CD3. Activated T cells treated with Hoechst 33342 (HO-33342) and analyzed by flow cytometry showed two major cell populations of high and low fluorescence. These populations were sorted and the DNA extracted and subjected to electrophoresis. Electrophoresis of DNA extracted from T cells showing a low level of HO-33342 fluorescence (HO-Low) resulted in a typical ladder pattern characteristic of internucleosomal DNA degradation associated with apoptosis, whereas the cellular DNA of the cells showing a high level of fluorescence (HO-High) showed a narrow high molecular weight band. Multiparameter analysis further indicated that cells with HO-High characteristics possessed corresponding high-FSC/low-SSC properties, whereas HO-Low cells formed a cluster of low-FSC/high-SSC cells. Analysis of the DNA extracted from cells sorted on the basis of scatter properties alone confirmed that the low-FSC/high-SSC population contained the apoptotic cells and that the high-FSC/low-SSC population was comprised of viable cells. This methodology allowed us to determine the percentage of apoptotic cells following anti-CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8+ cells in the cell cycle.

Animals↗
PubMed2006

The DNA polymerase activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts.

BACKGROUND: DNA polymerase epsilon (Pol epsilon) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol alpha and Pol delta in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol epsilon in chromosomal DNA replication is not well understood. RESULTS: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol epsilon. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol epsilon-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol epsilon holoenzyme to Pol epsilon-depleted extracts, but not by addition of polymerase-deficient forms of Pol epsilon, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol epsilon holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells. CONCLUSION: These results indicate that the DNA polymerase activity of Pol epsilon holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol epsilon holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.

Animals↗
PubMed1991

Deoxyribonucleic acid amplification and hybridisation in Crohn's disease using a chlamydial plasmid probe.

The possibility that Crohn's disease is caused by infection with Chlamydia trachomatis was examined by probing for chlamydial plasmid deoxyribonucleic acid (DNA) in DNA extracts from Crohn's disease tissue and by means of a serological study. Gut DNA extracts were obtained from 10 patients with Crohn's disease and four control subjects and were probed with a chlamydial plasmid probe after Southern blotting. The polymerase chain reaction was also used to amplify any chlamydial plasmid DNA present in tissue DNA extracts, before Southern blotting and probing. Chlamydial proctitis control specimens were not available: gut DNA extracts mixed with traces of chlamydia plasmid served as positive controls. Using these techniques, no chlamydial plasmid DNA sequences were found in Crohn's disease tissue. An enzyme linked immunosorbent assay for C trachomatis LI was performed on 48 patients with Crohn's disease and 48 control subjects. Seropositivity was present in 14.6% of patients and 29% of control subjects and was not statistically significant (p greater than 0.05). The failure to show chlamydial DNA and the lack of serological response to chlamydia make C trachomatis infection a very unlikely factor in the pathogenesis of Crohn's disease.

Adult↗
PubMed2005

STR-genotyping from human medieval tooth and bone samples.

We extracted the DNA contained in samples of bones and teeth from 10 skeletons excavated from the Gravette site (400-1000 AD, south of France). Ancient DNA was analysed by autosomal short tandem repeats (STRs). The DNA present in these ancient remains appeared very degraded, but nevertheless, better conserved in tooth than in bone samples. Moreover, we showed that the DNA extracted from ancient dental pulp was not exempt from polymerase chain reaction (PCR) inhibitors, which could result from extreme DNA fragmentation. An adapted protocol with a supplementary step of purification removed this inhibition.

Adult↗