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At least 973 records · Page 54Linked to original sources
PubMed1993

PCR jumping in clones of 30-million-year-old DNA fragments from amber preserved termites (Mastotermes electrodominicus).

DNA from 30-million-year-old amber preserved termites (Mastotermes electrodominicus) was PCR amplified with nuclear ribosomal RNA small subunit primers and cloned into the TA vector (INVITROGEN). We obtained several classes of recombinant clones as a result. Authentic Mastotermes electrodominicus clones were identified. The source of other classes of clones was identified as contaminants of the ancient DNA template. Several of the clones appeared to be chimeric in structure with half of the clone identical to the termite sequence and the other half identical to contaminant sequences. The phenomenon of PCR jumping was identified as a possible source for the chimeric clones.

Animals↗
PubMed1985

Isolation and characterization of DNA from Tritrichomonas foetus and Trichomonas vaginalis.

High molecular weight DNA samples free of contaminating proteins or RNA were obtained from Tritrichomonas foetus or Trichomonas vaginalis by lysing the cells in 4 M guanidinium thiocyanate before centrifuging in CsCl density gradient and then purifying the DNA band by NACS-37 column chromatography. The bulk DNA from either organism acted as a single component in ion-exchange chromatography, agarose gel electrophoresis, CsCl density gradient centrifugation and thermal denaturation. T. foetus DNA showed a melting temperature (Tm) of 82 degrees C corresponding to a 31% GC content whereas T. vaginalis DNA melted at 84 degrees C to suggest 36% GC. Both DNA samples demonstrated 35 to 42% hyperchromicity when fully melted. Cot analysis revealed the presence of repetitive sequences in both DNAs: approximately 46.7% in T. foetus DNA and 53.3% in T. vaginalis DNA. The unique sequences of these two protozoan DNAs are of a similar size of about 2.5 X 10(7) base pairs. Agarose gel electrophoresis of restriction fragments of the two purified DNA samples gave distinct banding patterns that were characteristic of the two species of protozoan parasites.

Animals↗
PubMed1993

Near-zero linking difference upon transcription factor IID binding to promoter DNA.

Binding of yeast transcription factor IID (TFIID) to the adenoviral major late promoter in circular DNA molecules caused a linking number change of less than 0.1. TFIID on its own therefore fails to unwind DNA appreciably, or else it causes both unwinding and compensatory writhing. Highly purified, recombinant yeast TFIID relaxed supercoiled DNA, because of a contaminant of bacterial topoisomerase I. Relaxing activity of topoisomerase I was enhanced by the adenoviral major late promoter, suggesting an instability of the TATA sequence or a destabilizing effect on flanking DNA.

Adenoviridae↗
PubMed2004

Elimination of locus-specific inter-individual variation in quantitative PCR.

Robust dosage PCR (RD-PCR), a duplex and quantitative PCR, detects large heterozygous deletions and duplications in genomic DNA samples. RD-PCR amplifies an endogenous internal control and a target locus. Two of six RD-PCR assays behaved anomalously due to lower yields specific to the targets. The variability was eliminated by heat treatment of the genomic DNA samples in 2x TE solution at 90 degrees C for 10 min. Heat treatment improves the utility of RD-PCR and may be generally helpful in multiplex PCR quantitation. The mechanism by which heat treatment eliminates inter-individual variation is unclear. The variability is not associated with DNA extraction methods, RNA contamination, or solution protein contamination, but may reflect inhibition from tightly bound chromatin proteins.

Artifacts↗
PubMed2000

[Contamination through preparation: risk of molecular genetic studies by using biological preservatives for museum collections].

In paleogenetic science, artifacts (i.e. non-authentic DNA sequences) are mainly produced by cryptic contamination with (i) edaphon DNA sequences and/or (ii) human biomolecules derived from the involved researchers and the laboratory equipment. A third, and yet underestimated source of contamination with exogenous nucleic acids is provided by (iii) conservation practices applied to old material. Bone glue has been successfully used from the beginning of the 19th century up to the middle of this century, and comprises a rich source of non-authentic nucleic acids. An unequivocal identification of treated samples remains difficult since bone and the glue used for conservatory purposes bear similar chemical properties. Since the majority of agents used for the preservation of museum collections are of biological origin, the differentiation between contaminated and non-treated samples is required.

Artifacts↗
PubMed2003

Spermine compaction is an efficient and economical method of producing vaccination-grade DNA.

Plasmid DNA inoculations can induce both humoral and cellular immunity, and this technique is now being employed in developing vaccination regimens for a large number of applications. DNA vaccination studies require the preparation of large amounts of purified plasmid DNA with low endotoxin contamination, and the cost burden for multiple injections, multiple animal or large animal studies is significant. We recently reported that selective compaction with spermine can be used to purify large quantities of DNA. We wanted to determine whether this method would produce DNA suitable for vaccination. Endotoxin levels for spermine-compacted DNA were 0.3+/-0.01 endotoxin units (EU)/microg, well within the accepted range (less than 3 EU/microg) for in vivo use. When injected intramuscularly into mice, column-purified and spermine-compacted DNA induced an equivalent antigen-specific CD8+ T-cell response. The labor and time involved in purifying 5 mg of DNA by each method were similar, but the cost of spermine-compacted DNA was only 20% of the cost of column-purified DNA. We conclude that spermine compaction is an efficient and economical method for preparing vaccination-grade DNA.

Animals↗
PubMed2006

Analysis of one million base pairs of Neanderthal DNA.

Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.

Animals↗
PubMed1993

DNA typing of urine samples following several years of storage.

The possibility of typing DNA polymorphisms on urine samples was investigated in a controlled storage experiment and for samples that were 1-7 years old. Female urine samples showed a higher amount of epithelial cells and therefore a higher DNA yield. Employing the polymerase chain reaction, specific amplification results were achieved for all samples over a 6 month storage period. The microscopical examination of the samples revealed not only differing degrees of contamination with bacteria, yeasts and fungi, but also the presence of still intact epithelial cells. Only 20% of the male samples and 32% of the female samples yielded specific amplification results. By separating the human cells from the contaminating organisms prior to DNA extraction, the number of successfully typed samples could be improved to 35% of the male and 77% of the female samples. This result confirms that excess amounts of co-extracted non-human DNA can inhibit the specific amplification of human target sequences.

Cell Count↗
PubMed1993

Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction.

We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.

Base Sequence↗
PubMed1977

Binding of the partially purified glucocorticoid receptor of rat liver to chromatin and DNA.

The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [3H]-triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases. If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes. The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a 10-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the "accessible" DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations.

Animals↗
PubMed1994

Respiratory contamination of polymerase chain reactions by human herpesvirus 6.

Human herpesvirus type 6 DNA derived from human breath was discovered to contaminate PCR reactions during routine reaction preparation. Parallel PCR experiments were conducted in which expiratory secretions were blocked by a surgical mask, while others were performed without any attempt to circumvent respiratory contamination. The experimenter was previously determined to harbor HHV-6 DNA in the saliva. All reactions in which expiration was obstructed were negative for HHV-6 DNA via PCR. Reactions in which there was no attempt to obstruct respiratory secretions were positive for HHV-6 DNA. These data suggest that PCR assays investigating the presence of HHV-6 may be highly susceptible to contamination from the experimenter leading to false positive results.

Aerosols↗
PubMed1985

An economical large scale procedure to purify E. coli amplifiable plasmids for DNA sequencing, in vitro transcription and in vitro mutagenesis.

A reproducible and economical procedure for obtaining a large and quantitative yield of highly purified covalently closed circular plasmid DNA is described. The procedure departs in several ways from more commonly used methods. These are a) avoidance of the use of CsCl, ethidium bromide and ultracentrifuge, b) enrichment of the plasmid DNA by selective denaturation of chromosomal DNA with an alkaline-SDS solution, c) enrichment of covalently closed circular plasmid DNA by extraction with acid-phenol, and d) removal of small degraded RNA fragments by molecular sieve chromatography after digestion with RNase A. The plasmid DNA prepared by this new procedure is free of contaminants and has been used for DNA sequencing, in vitro transcription, transformation and in vitro mutagenesis.

Base Sequence↗
PubMed2005

Monitoring Arthrobacter protophormiae RKJ100 in a 'tag and chase' method during p-nitrophenol bio-remediation in soil microcosms.

Monitoring of micro-organisms released deliberately into the environment is essential to assess their movement during the bio-remediation process. During the last few years, DNA-based genetic methods have emerged as the preferred method for such monitoring; however, their use is restricted in cases where organisms used for bio-remediation are not well characterized or where the public domain databases do not provide sufficient information regarding their sequence. For monitoring of such micro-organisms, alternate approaches have to be undertaken. In this study, we have specifically monitored a p-nitrophenol (PNP)-degrading organism, Arthrobacter protophormiae RKJ100, using molecular methods during PNP degradation in soil microcosm. Cells were tagged with a transposon-based foreign DNA sequence prior to their introduction into PNP-contaminated microcosms. Later, this artificially introduced DNA sequence was PCR-amplified to distinguish the bio-augmented organism from the indigenous microflora during PNP bio-remediation.

Arthrobacter↗
PubMed2003

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.

DNA↗
PubMed2005

Contaminants of PC-SPES are not responsible for cytotoxicity in human small-cell lung carcinoma cells.

PC-SPES is a mixture of herbs used in the treatment of prostate cancer. Batches of this product were found to contain traces of synthetic drugs, and the product was removed from the market. On the basis of a correlation between contaminant levels and cytotoxicity in prostate carcinoma cell lines, Sovak et al. [M. Sovak, A. Seligson, M. Konas, M. Hajduch, M. Dolezal, M. Machala, R. Nagourney, Herbal composition PC-SPES for management of prostate cancer: identification of active principles, J. Natl Cancer Inst. 94 (2002) 1275-1281] concluded that the contaminants were responsible for cytotoxicity of this preparation. Previously, we showed that extracts of PC-SPES are cytotoxic and pro-apoptotic in both drug-sensitive (H69) and drug resistant (H69V) human small-cell lung carcinoma (SCLC) cell lines. Here, we investigated whether the contaminants might be responsible for these effects. In contrast to the data reported for prostate carcinoma cells, extracts of batches of PC-SPES from the year 1998 (reportedly contaminated) and 2001 (much less contaminated) were equally cytotoxic in both SCLC cell lines. Tests of individual contaminants gave IC50 values far in excess of the amounts reported to be present in the IC50 level for the PC-SPES extracts: diethlystilbesterol: actual IC50 in H69 cells, >1000 microM; concentration present in herbal extract at IC50, 0.05-0.2 microM; indomethacin: actual IC50 in H69 cells, 800 microM; concentration in herbal extract, 1.5-20 microM; warfarin: actual IC50 in H69 cells, 950 microM; concentration in herbal extract, 0.57-0.93 microM. Adding the calculated maximum concentration of the contaminants, singly or in combination, to extracts of the less contaminated batch (2001) of PC-SPES did not alter the cytotoxicity of the extract in H69 or H69V cells. At the contaminated concentrations, as well as 5 x those concentrations, none of the contaminants was pro-apoptotic, as indicated by a DNA fragmentation kinetics assay. However, extracts of both early and late batches of PC-SPES were pro-apoptotic in SCLC cells. We conclude that the traces of pharmaceuticals found in PC-SPES were not responsible for its cytotoxic and pro-apoptotic activities of this herbal mixture on SCLC cells.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Estimation of bacterial contamination in ultrapure water: application of the anti-DNA antibody.

We constructed and established a hybridoma cell line that produces immunoglobulin G-type anti-DNA antibody. By using this antibody, we could successfully detect a single bacterial cell in ultrapure water (UPW). The detection system is composed of a membrane-supported western blotting-type immunoassay and a two-dimensional photon analyzer with high resolution. It can detect and count every bacterial cell in a wide field of view on a trapping filter i.e., a circle with an 18-mm diameter. This means 10 fg (10(-14) g) of bacterial DNA can be detected in the field. This system could be a useful tool for evaluating the number of bacteria contained by UPW and water used for medical purposes.

Animals↗