SEARCH · PubMed Health

Results for “DNA Contamination”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources

PubMed2005

Mitochondrial DNA haplotyping revealed the presence of mixed up benign and neoplastic tissue sections from two individuals on the same prostatic biopsy slide.

DNA typing was requested to investigate a presumptive cancer diagnosis error by confirming whether benign and cancerous prostatic tissue in the same presurgical haematoxylin and eosin stained slide belonged to the same person. After independent histological re-examination of the slide by a pathologist, manual slide dissection was used to guarantee independent and high recovery DNA isolation from each tissue section, avoiding carryover and background contamination. Nuclear DNA quantification performed by real time polymerase chain reaction (PCR) revealed the absence of human DNA for short tandem repeat (STR) typing. Mitochondrial DNA was only obtained by performing PCR of very short fragments ( approximately 100 bp), indicating high DNA degradation. Different low frequency hypervariable region I haplotypes were obtained from each tissue section (normal tissue section haplotype: 16224C, 16234T, 16311C, 16356C; cancer tissue section haplotype: 16256T, 16270T, 16293G). Only the normal tissue section haplotype matched that obtained from the patient's blood sample, indicating that the cancer tissue section originated from an unknown patient. These results supported the hypothesis of sample mix up during block processing or slide preparation by a carryover mechanism. Mitochondrial genetic typing is recommended to exclude the possibility of carryover artefacts when low DNA content and high degradation compromise conventional STR typing.

Artifacts↗

PubMed1975

Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells.

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.

Animals↗

PubMed1998

The construction and use of a PCR internal control.

An example of the application and contruction of a polymerase chain reaction (PCR) internal control is presented. The internal control is synthesized in one PCR reaction. The primers used in this reaction possess 5' over-hanging ends which are identical to the primers used in the diagnostic reaction, whereas their 3' ends are complementary to a predetermined DNA sequence (pUC19 in this case) of defined length and sequence. As the sequence of the control except for primer sites, is not homologous to the PCR signal product, the formation of heteroduplexes and non-specific PCR products should not occur. Neither is there a risk that the target DNA will contaminate the internal control. However, the simultaneous amplification of two different DNA fragments flanked by the same primer sites resulted in either inhibition or enhancement of one or both products depending on the molar ratio of those DNA fragments. The presented method may be applied to construction of internal controls for quantitative PCR. The internal control was developed and tested for use in a PCR detection system for Agrobacterium tumefaciens.

Agrobacterium tumefaciens↗

PubMed1999

Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.

Drug Resistance, Microbial↗

PubMed1991

Purification of mitochondrial DNA from total cellular DNA of small tissue samples.

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.

Animals↗

PubMed1999

Active human hepatitis B viral polymerase expressed in rabbit reticulocyte lysate system.

Human HBV polymerase has been expressed in reticulocyte lysate system. The expressed protein shows the DNA-dependent DNA polymerase activity. In vitro transcription and translation produces a major protein product with an apparent molecular weight of approximately 100 kD. The HBV DNA polymerase has been characterized biochemically in the condition that the contaminating cellular DNA polymerases were fairly suppressed by aphidicolin and NEM. The polymerization reaction is optimal at pH 7.5 and 37 degrees C and the polymerase requires either MnCl2 or MgCl2, with a preference for MnCl2. The protein represented an optimal activity in the presence of either 75 mM NaCl or 100 mM KCl, with a higher activity at 75 mM NaCl than 100 mM KCl. Study of the polymerizing activity of the deleted versions of the polymerase protein suggests that the terminal protein is essential for full polymerase function and the spacer region may decrease the stability of the P protein.

Animals↗

PubMedDate not supplied

Limited selection of sodium channel blocking toxin-producing bacteria from paralytic shellfish toxin-contaminated mussels (Aulacomya ater).

Paralytic shellfish toxins (PSTs) are sodium channel blocking (SCB) toxins, produced by cyanobacteria, as well as by marine dinoflagellates and their associated bacteria, and cause serious health and economic concern worldwide. In a previous study, approximately 70% of the bacteria enriched from PST-contaminated shellfish tissue and isolated on marine agar medium were observed to produce SCB toxins. In the study reported here, the high percentage of cultivable toxigenic bacteria is demonstrated to be obtained through a marked selection on marine agar medium. The cultivable as well as the total bacterial diversity associated with PST-contaminated shellfish collected from the Magallanes region in the south of Chile has been analysed. Approximately 80% of bacterial isolates, analysed by restriction analysis of PCR amplified ribosomal DNA (i.e., ARDRA fingerprinting), were limited to only two genotypic OTUs (operational taxonomic unit). Sequence determination and analysis of the 16S rDNA from representative isolates of both OTUs established them to be closely related to species of the Psychrobacter genus of the gamma-subclass of the Proteobacteria. The total bacterial diversity in the shellfish was further analysed, using a cultivation-independent strategy of extraction of total DNA from contaminated tissue, PCR-amplification of bacterial 16S rRNA genes, cloning of the PCR products and analysis of the cloned 16S rDNA sequence types by fingerprinting and sequencing. Only 2% of the cloned sequence types corresponded to species of the Psychrobacter genus. The 16S rDNA sequence types detected clustered with species of the y-Proteobacteria subclass, the Cytophaga-Flexibacter-Bacteroides (CFB), the Fusobacteria and the Firmicutes phyla. The level of diversity observed within the libraries of cloned 16S rDNA was markedly greater than that observed among isolates obtained through marine agar enrichment cultures from the same shellfish tissue. Additionally the predominant cloned 16S rDNA sequence types detected from samples of the surrounding seawater demonstrated no correlation with those observed in the PST-contaminated mussels.

Animals↗

PubMed1994

DNA adducts of the ubiquitous environmental contaminant cyclopenta[cd]pyrene.

The bioactivation of cyclopenta[cd]pyrene (CPP) was investigated to determine the major DNA adduct-forming metabolite(s) of this widespread environmental contaminant and suspect carcinogen. DNA adducts were analyzed by 32P-postlabeling. Four major and at least seven minor adducts formed when CPP was incubated with calf thymus DNA in the presence of rat liver microsomal systems. P450 subfamilies IA and IIB both activated CPP as microsomes from either phenobarbital- or beta-naphthoflavone-treated rats produced quantitatively similar and qualitatively identical adducts. When the epoxide hydrolase inhibitors, 1,1,1-trichloropropene-2,3-oxide or cyclohexene oxide were added to the incubations, binding increased 2.5- to 4-fold, suggesting epoxidation as a mechanism of adduct formation in vitro. Sprague-Dawley rats were killed 1, 3, 7, 18, 45 and 80 days postdosing i.p. with 50 mg/kg CPP. In all tissues analyzed, four major and several minor qualitatively identical adducts were produced. Binding was highest and most persistent in lung followed by heart, white blood cells (WBCs) and liver. CPP adducts were detectable at doses from 1 microgram/kg to 50 mg/kg. Rat lung DNA adducts were cochromatographed with standardized deoxyguanosine and deoxyadenosine adducts produced by reaction of CPP-3,4-epoxide in vitro. All rat lung adducts comigrated with the deoxyguanosine adducts but one was clearly deoxyadenosine derived. Mouse skin DNA adducts from NIH Swiss mice and mouse lung DNA adducts from B6C3F1 mice were also analyzed. All adducts from either mouse tissue comigrated with rat lung DNA adducts, suggesting CPP-3,4-epoxide was also the major DNA adduct-forming species in the mouse. CPP-3,4-epoxide has been suggested to be the key mediator of the biological activities of CPP. Evidence presented here strongly suggests CPP-3,4-epoxide as the major adduct-forming species of CPP as catalyzed in vitro by rat liver preparations known to mediate the mutagenic activation of CPP, in the rat in vivo, and in mouse skin and lung, two tissues with known sensitivity to CPP tumorigenicity.

Animals↗

PubMed2006

Specific detection of residual CHO host cell DNA by real-time PCR.

Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format. Real-time PCR primers were designed to amplify a 150bp region of a genomic fragment from hamster DNA. The specificity of the probe was evaluated in real-time PCR reactions using genomic DNA from mouse fibroblast, human kidney and hamster ovary cell lines as template. Sensitivity of real-time PCR was compared on genomic DNA from hamster cell line CHO DG44. These primers can be used in real-time PCR reactions to detect presence of contaminating hamster DNA in purified protein samples down to sensitivity of 300fg genomic DNA.

Animals↗

PubMed2002

Reduction of the rate of false-positive cultures of Mycobacterium tuberculosis in a laboratory with a high culture positivity rate.

Our laboratory, engaged in a prospective study of adult pulmonary tuberculosis, processed on average 1186 sputum samples per year for the detection of Mycobacterium tuberculosis (M. tuberculosis). Approximately 55% of all sputum samples were culture-positive. The study protocol required that all patients had their M. tuberculosis isolates DNA fingerprinted at diagnosis, and at subsequent time points if the patients either failed treatment or presented again with tuberculosis. Over a 22-month period, there were 14 apparent treatment failures from 109 patients who had completed 6 months of therapy. Only two of these were true treatment failures, while the other 12 had DNA fingerprints that were different from those obtained at diagnosis. It was concluded that these 12 cultures represented episodes of laboratory cross-contamination. Retrospective DNA fingerprinting of patient isolates was done so that each patient had at least two independent isolates fingerprinted. This survey revealed that 7.3% of DNA fingerprints were discordant. False-positive cultures with discordant DNA fingerprints generally arose late in chemotherapy and the isolates were usually co-processed with other strongly smear-positive sputum samples. Simple modifications of laboratory procedures were made, and over a following 10.5-month period the false-positive rate was reduced to 2.1%. These modifications did not increase the workload or the cost of processing samples and can thus be used successfully by any laboratory, and particularly by those in resource-poor settings.

Adult↗

PubMed2005

Contamination with hepatitis B virus DNA in gastrointestinal endoscope channels: risk of infection on reuse after on-site cleaning.

BACKGROUND AND STUDY AIMS: The incidence of viral contamination in the air, water and suction/accessory channels of gastrointestinal endoscopes was examined in order to evaluate the risk of infection. MATERIALS AND METHODS: After endoscopic examinations, including biopsy procedures, in 17 patients who were positive for hepatitis B virus surface antigen and eight patients who were positive for hepatitis C virus antibody, the endoscopes were cleaned on site by suctioning and flushing the air and water channels with an enzyme detergent. First samples were then collected by flushing 5 ml of sterile water through each channel. After mechanical reprocessing, second samples were collected in the same way. Virological studies were carried out with real-time polymerase chain reactions for hepatitis B virus DNA and hepatitis C virus RNA. RESULTS: Hepatitis B virus DNA was detected in five of the first samples recovered from the suction/accessory channels of the endoscopes (titers of 1.3 x 10 (4) to 2.5 x 10 (5) copies/ml), while no contamination was detected after reprocessing ( P = 0.0445). The first samples from one water channel and three air channels were also positive for hepatitis B virus DNA, but were negative after reprocessing ( P > 0.5, P = 0.227, respectively). No hepatitis C virus RNA was detected in any of the samples. CONCLUSIONS: These results indicate that all of the channels were potential sources of viral infection.

Biopsy↗

PubMed1999

Gene therapy in transplantation: pathological consequences of unavoidable plasmid contamination with lipopolysaccharide.

Experimental studies evaluated the responses of murine cardiac graft recipients to high and low levels of lipopolysaccharide (LPS) contaminating plasmid DNA preparations. Immediately prior to transplantation, graft recipients were transfected by injecting the quadriceps muscles with plasmids that encoded the murine interleukin (IL)-4 gene and beta-galactosidase (beta-gal) gene. Graft recipients transfected with plasmids encoding only the beta-gal gene served as negative plasmid controls. Three groups of mice were transfected with plasmids containing high levels of contaminating LPS: (a) nontransplanted C57B1/6 mice, (b) C57B1/6 cardiac isograft recipients, (c) DBA/2 (H-2d)-->C57BL/6 (H-2b) cardiac allograft recipients. Unexpectedly, graft failure within 24 h was observed in IL-4 transfected isograft and allograft recipients, but not in mice transfected with the beta-gal gene alone. However, histopathological findings, for example, vascular cell adhesion moelcule-1 (VCAM-1) expression in cardiac grafts and mononuclear lung infiltration, were remarkably similar for both treatment groups and consistent with LPS-induced pathology. LPS assays were used to evaluate four different methods of plasmid purification for degree of LPS contamination. A successful strategy for reducing levels of LPS contamination was identified and transfection experiments repeated in cardiac allograft recipients receiving LPS inoculum that were minimized and standardized (6.4 EU/mouse) for all treatment groups. Despite receiving substantially lower levels of LPS, in all treatment groups there was persistent cardiac graft endothelial cell activation manifested by VCAM-1 expression and persistent, albeit less severe, lung pathology. We found that plasmid contamination with LPS was unavoidable and that even very low levels can alter immune responses in transplant recipients confounding data interpretation. Thus, it is imperative to account for LPS contamination in experiments utilizing plasmid DNA for gene transfer, especially in experimental models of immunity and inflammation.

3T3 Cells↗

PubMed1993

Theileria parva: detection of genomic polymorphisms by polymerase chain reaction amplification of DNA using arbitrary primers.

Genomic DNA fingerprints were generated from Theileria parva piroplasm DNA by polymerase chain reaction amplification using arbitrary decamers as primers. Four of 12 primers tested generated amplified fragments which were polymorphic between different T. parva stocks or clones. Hybridization of radiolabeled total amplification products to Southern blots of T. parva piroplasm and uninfected bovine DNA confirmed that none of the amplified fragments were derived from any contaminating bovine DNA which may have been present in small quantities. Polymorphic DNA fragments were reproducibly amplified from independent DNA preparations made from cloned parasites. Individual amplified DNA fragments were gel purified and shown to hybridize to size-polymorphic EcoRI fragments in T. parva genomic DNA. Hybridization of individual amplified fragments to total fingerprints, generated using the same arbitrary primer, demonstrated that a single primer detected at least three independent genomic polymorphisms in T. parva DNA.

Animals↗

PubMed1993

Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays.

A simple method for the production of internal control DNA for two well-established Mycobacterium tuberculosis polymerase chain reaction assays is described. The internal controls were produced from Mycobacterium kansasii DNA with the same primers but at a lower annealing temperature than that used in the standard assays. In both assays, therefore, the internal control DNA has the same primer-binding sequences at the target DNA. One-microgram quantities of internal control DNA which was not contaminated with target DNA could easily be produced by this method. The inclusion of the internal control in the reaction mixture did not affect the efficiency of amplification of the target DNA. The method is simple and rapid and should be adaptable to most M. tuberculosis polymerase chain reaction assays.

DNA, Bacterial↗

PubMed1993

Solid phase purification in automated DNA sequencing.

A solid phase purification method has been utilized to investigate the effect of template DNA and other contaminants present in enzymatic DNA sequencing reactions upon data quality in automated DNA sequence analysis. The primer oligonucleotide is prepared with an internal biotin group and one of four fluorescent tags at the 5' terminus. The fluorescently labeled DNA fragments produced in the sequencing reactions are separated from other reaction components, including template DNA, using superparamagnetic streptavidin beads. Template DNA and other reaction components may then be added back to the purified material to assess their effect upon sequence data. It is found that band intensity and base calling accuracy are decreased by the presence of the template DNA and other reaction components.

Animals↗

PubMed1989

DNA extraction from bloodstains in respect to age and stained substrate.

The amount and the quality of DNA that could be extracted from bloodstains equivalent to 200 microliters blood were examined after 3, 6, 9, 12, 15, 18 and 21 days of storage under dry or humid conditions at room temperature. DNA was also extracted from two days old 200 microliters bloodstains on different stain carriers. The amount of DNA that could be extracted showed a dependency on all the parameters that were examined. On carriers with a rough surface structure, where the blood can soak in, the resolving of leukocytes was impaired and the DNA recovery rate was low, e.g. carpeting and suede. The DNA yield was higher for substrates with a smooth surface, e.g. paper, glass and smooth leather. The aging experiments revealed that for the stains stored under dry conditions, there was no decrease in the DNA yield for stains on glass, while the amount of DNA that could be extracted became less for older stains on cotton. Under humid conditions the DNA yield was high and did not decrease for both glass and cotton fabric. The quality of the DNA was not impaired by aging or storage conditions, but for several stain carriers the DNA was contaminated by chemical substances which inhibited restriction enzyme digestion and successful DNA-typing.

Aging↗

PubMed1985

Early events in duck hepatitis B virus infection. Sequential appearance of viral deoxyribonucleic acid in the liver, pancreas, kidney, and spleen.

Early events in duck hepatitis B virus infection were studied in 1-day-old ducklings following inoculation. Group A ducklings (n = 26) were inoculated intraperitoneally with 10 microliter of infective serum, and group B ducklings (n = 29) were inoculated with 50 microliter. Samples of the serum, liver, pancreas, kidney, and spleen were taken, starting 3 h after inoculation and continuing through the 14th day. In group A, relaxed circular double-stranded deoxyribonucleic acid (DNA) did not appear in serum until day 10, whereas single-stranded DNA, indicative of active replication of the virus, was already demonstrable in the liver on day 6. In group B, single-stranded DNA was first detected in the liver on day 3, and relaxed circular double-stranded DNA became detectable in the liver and serum on day 6. The pancreas started to have single-stranded DNA on day 10 in group A and on day 6 in group B, suggesting active viral replication in this organ soon after it occurred in the liver. In the spleen, relaxed circular double-stranded DNA was detectable when serum became positive for viral DNA, probably due to contamination by serum DNA. However, single-stranded DNA became detectable on day 14 in group A and on day 6 in group B, suggesting a delayed but active viral replication in the constituent tissues of the spleen. These results have demonstrated that active replication of duck hepatitis B virus starts in the liver after infection, and is followed by the pancreas, the kidney, and the spleen. The incubation period is shortened when larger amounts of virus are inoculated, but the sequential occurrence of viral replication in these organs remains the same.

Animals↗

PubMed2004

The bacterial catalase from filarial DNA preparations derives from common pseudomonad contaminants and not from Wolbachia endosymbionts.

Wolbachia are obligatory endosymbionts in many species of filarial nematodes. Certain bacterial molecules induce antibody responses in mammalian hosts infected with filariae, while others activate inflammatory responses that contribute to pathology. These findings, coupled with antibiotic studies demonstrating the dependence of filarial embryogenesis on the presence of Wolbachia, have intensified research on Wolbachia-nematode interactions, and the effects of Wolbachia molecules on the mammalian immune system. By amplification and sequencing of 16S rDNA and catalase sequences, we show that filarial DNA samples prepared from nematodes collected under typical conditions are frequently contaminated with Pseudomonas DNA. Analysis of a published DNA fragment containing a catalase attributed to the Wolbachia of Onchocerca volvulus showed it to be most like Pseudomonas, both in terms of sequence similarity and genomic organization. Additionally, there was no obvious catalase in either of two available Wolbachia genome sequences. Contamination of filarial DNA with bacterial sequences other than Wolbachia can complicate studies of the role of these symbionts in filarial biology.

Animals↗