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Results for “DNA Contamination”

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At least 109 records · Page 6Linked to original sources

PubMed1998

Molecular epizootiology of genotoxic events in marine fish: linking contaminant exposure, DNA damage, and tissue-level alterations.

Molecular epizootiological studies are increasingly being used to investigate environmental effects of genotoxic contaminants. The assessment of damage to DNA and linking the damage to subsequent molecular, cellular, or tissue-level alterations is a central component of such studies. Our research has focused on the refinement of the 32P-postlabeling assay for measuring covalent DNA-xenobiotic adducts arising from exposure to polycyclic aromatic compounds, using DNA adducts as molecular dosimeters of genotoxic contaminant exposure in biomonitoring studies, and investigating the relationship of DNA adduct formation to toxicopathic liver disease, including neoplastic lesions. A combination of field and laboratory studies using the 32P-postlabeling assay has shown that DNA adducts in marine fish are effective molecular dosimeters of genotoxic contaminant exposure. Investigations of the relationship of DNA adduct formation to neoplastic liver disease have shown that elevated levels of DNA adducts in certain fish species from contaminated coastal sites are associated with increased prevalences of toxicopathic hepatic lesions, including neoplasms, and that the ability to assess DNA damage has helped to explain, in part, species differences in lesion prevalence. Moreover, in a study of a site in Puget Sound contaminated with polycyclic aromatic compounds, we have shown, for the first time, that elevated levels of hepatic DNA adducts are a significant risk factor for certain degenerative and preneoplastic lesions occurring early in the histogenesis of hepatic neoplasms in feral English sole (Pleuronectes vetulus). These latter findings coupled with our current studies of mutational events in the K-ras proto-oncogene should provide further mechanistic substantiation that mutagenic events resulting from exposure to complex mixtures of genotoxic polycyclic aromatic compounds are involved in the etiology of hepatic neoplasia in English sole.

Animals↗

PubMed2006

[Effect of environmental contaminants on DNA methyltransferase activity of mouse preimplantation embryos].

OBJECTIVE: To investigate the effect of environmental contaminants on development rate and DNA methyltransferase activity of preimplantation embryos. METHODS: 1-cell embryos were collected from oviduct, in vitro cultured to blastocyst stage in media with or without environmental contaminants. The development rate and DNA methyltransferase activity were determined. RESULTS: The development rates of preimplantation embryos from 1-cell stage to blastocyst stage were not affected by the exposed compounds. However, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) significantly increased the DNA methyltransferase activity, 2, 2', 3, 3' 4, 4'-polychlorinated biphenyl (PCB153) and diethylstilbestrol (DES) significantly decreased the DNA methyltransferase activity, while p, p'- dichlorodiphenel ethylene (DDE) and dibutyl phthalate (DBP) did not change the DNA methyltransferase activity significantly. CONCLUSION: Exposure to TCDD, DES, and PCB 153 alters the DNA methyltransferase activity in early development stage, which may induce the change of methylation status of genes and affect the development in the later stage.

Animals↗

PubMed2000

Poly(A) cDNA-specific (PACS) RT-PCR: a quantitative method for the measurement of any poly(A)-containing mRNA not affected by contaminating genomic DNA.

We present a simple and efficient RT-PCR method for the detection and quantitation of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA and does not rely on exhaustive DNase digestion protocols. The technique described here requires the use of an antisense primer designed to contain 6-8 bp cDNA-specific sequence and an additional 17 Ts located on the 5' end to take advantage of the poly(A) tail. A second cDNA-specific sense primer can be used that does not need to be separated by intronic DNA sequence.

Animals↗

PubMed1992

[Rapid prenatal diagnosis of cystic fibrosis using the polymerase chain reaction: results of the first 5 cases].

Cystic fibrosis (CF) is an autosomal recessive lethal disease with an incidence in Slovakia of 1 affected in 1800 newborns. Within a year the incidence amounts to about 50 cases. Though the responsible gene has already been cloned, the only effective approach to prevention is prenatal diagnosis in the first and second trimester of pregnancy. The paper presents the results of the first five cases of prenatal diagnosis of CF established by the new rapid method of DNA analysis, polymerase chain reaction (PCR). Delta F508 deletion mutation and closely linked DNA polymorphism KM19/PstI were assessed. In two of the five cases studied the fetuses were found to be affected and pregnancy termination was indicated. To exclude the possibility of fetal DNA contamination with maternal DNA, the hypervariable DNA polymorphism VNTR apoB was determined simultaneously. The advantages of this approach are demonstrated on cases of prenatal diagnosis performed in two families where contamination of fetal DNA could be excluded. The value of the PCR method is being compared with that of Southern's hybridization method. (Tab. 2, Fig. 4, Ref. 27.).

Cystic Fibrosis↗

PubMed2004

Immobilized cobalt affinity chromatography provides a novel, efficient method for herpes simplex virus type 1 gene vector purification.

Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early-phase clinical trials, emphasizing the need for high-yield, scalable manufacturing processes that result in virus preparations that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu2+, Zn2+, Ni2+, and Co2+) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA.

Adsorption↗

PubMed1999

Identification of contaminating fungal DNA sequences in Zymolyase.

When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the Saccharomyces sensu stricto complex and Kluyveromyces lodderae. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.

DNA Primers↗

PubMed1998

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling.

32P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 microM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched 32P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compared with the phenol-extraction (14.3 +/- 0.8). RNA was approximately 4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P-postlabeling.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1991

Solid phase capture method for the specific amplification of microbial nucleic acids--avoidance of false-positive and false-negative reactions.

DNA amplification assays such as the polymerase chain reaction are being developed for the amplification of small quantities of microbial nucleic acids. These assays offer the potential for a great deal of sensitivity. However, the high level of sensitivity increases the likelihood of cross-contamination of amplified products and the generation of false-positive reactions. In addition, substances in body fluids can inhibit the efficient performance of the amplification reactions. We have developed an assay format in which microbial nucleic acids are specifically bound to a solid phase surface. Contaminating DNA and enzyme inhibitors present in the sample are removed by washing prior to the performance of the amplification reaction. We could use this system to amplify and detect small amounts of HIV DNA diluted in whole blood. The assay system could distinguish target DNA from contaminating DNA fragments generated by prior amplification reactions.

Base Sequence↗

PubMed1999

Proving the authenticity of ancient DNA by comparative genomic hybridization.

In PCR-supported amplification of ancient, degraded DNA, contamination with contemporary DNA can lead to false-positive results, which frequently give rise to discussions in which the mere existence of ancient DNA is doubted. Our confirmation of ancient DNA using comparative genome hybridization (CGH) eliminates these doubts. Unlike PCR methods, CGH requires no amplification of the DNA to be analyzed if adequate amounts of specimen DNA is used. Thus, false results traceable to contaminations are practically ruled out. The examples provided here prove the authenticity of ancient DNA for a 250-year-old and a 3,000-year-old sample. At the same time, the CGH of ancient DNA offers the chance to gain insight into the pattern of DNA degradation and to monitor the preservation of certain chromosomal segments.

Chromosomes, Human↗

PubMed2004

The inhibitory effect of pentobarbitone on reverse transcription-PCR.

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.

Animals↗

PubMed2006

Tracking down human contamination in ancient human teeth.

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.

DNA, Mitochondrial↗

PubMed2003

Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates.

A real-time PCR assay was developed for rapid detection of eubacterial 16S ribosomal DNA in platelet concentrates. The sensitivity of this assay can be hampered by contaminating DNA in the PCR reagents. Digestion of the PCR reagents with Sau3AI prior to PCR amplification was effective in eliminating this contaminating DNA without affecting the sensitivity of the assay.

Blood Platelets↗

PubMedDate not supplied

P450 aromatase messenger ribonucleic acid expression in male rat germ cells: detection by reverse transcription-polymerase chain reaction amplification.

We have previously demonstrated that cytochrome P450 aromatase (P450arom) protein, an estrogen-synthesizing enzyme, is present and active in germ cells of the adult mouse testis. To establish that P450arom mRNA is expressed in germ cells of other species, we examined expression of P450arom in adult rat germ cells by employing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from Staput separated germ cells and reverse transcribed. The resulting cDNA was amplified by nested PCR reactions using oligonucleotide primers selected from a highly conserved region of the P450arom gene. RT-PCR analysis yielded cDNA products of 334 bp in length that corresponded to the predicted size expected from the final nested amplification. The identity of the germ cell P450arom PCR products was confirmed by restriction enzyme analysis and direct nucleotide sequencing. Rat genomic DNA was subjected to PCR to verify that P450arom DNA products were not obtained from genomic DNA contamination. Rat genomic DNA yielded a nested PCR product for P450arom of approximately 2000 bp, suggesting that, as is the case with the human P450arom gene, the rat P450arom gene contains an intron in the amplified region. In addition, a semiquantitative technique was utilized to eliminate the possibility that the P450arom RT-PCR products were derived from Leydig cell contamination of Sta-put-separated germ cell preparations. RT-PCR for P450arom and 3-beta-hydroxysteroid dehydrogenase (3 beta-HSD), a Leydig cell-specific steroidogenic enzyme, was carried out on Sta-put-separated germ cells and interstitial cell preparations containing Leydig cells. P450arom and 3 beta-HSD RT-PCR reactions were stopped at three cycle intervals to detect and compare the earliest appearance of RT-PCR reaction products in various cell types. Results indicated that P450arom mRNA is detected in round spermatids before it is detected in interstitial cells, whereas 3 beta-HSD was detected only in interstitial cells, suggesting that the P450arom mRNA detected in germ cells is not due to interstitial cell contamination of germ cell preparations. Therefore, our results indicate that P450arom mRNA is expressed in adult rat germ cells and that testicular germ cells are a potential source of estrogen in the male reproductive tract.

Amino Acid Sequence↗

PubMed1995

Insertions and duplications of mtDNA in the nuclear genomes of Old World monkeys and hominoids.

Using oligonucleotide primers designed to match conserved regions of mammalian mitochondrial DNA (mtDNA), we have amplified and sequenced two divergent cytochrome b nuclear pseudogenes from orangutan cellular DNA. Evolutionary analysis suggests that a nuclear transfer occurred about 30 million years ago on the lineage leading to the catarrhines (Old World monkeys and hominoids), and involved a long (at least 3 kilobases), probably damaged, piece of mtDNA. After this transfer, the pseudogene duplicated, giving rise to the two copies that are probably present in all hominoids, including humans. More recent transfers involving the entire cytochrome b gene have also occurred in the Old World monkeys. Such nuclear copies of mtDNA can confound phylogenetic and population genetic studies, and be an insidious source of DNA contamination of 'ancient' and forensic DNA. Indeed, contamination with these anciently transferred human pseudogenes is almost certainly the source of the cytochrome b sequences recently reported from 'dinosaur bone DNA'.

Animals↗

PubMed1990

'DNA' as contaminants in antibiotics and its capacity to transform bacteria to drug resistance.

DNA/and deoxyribose sugars were detected in streptomycin (Sm), kanamycin, polymyxin, penicillin G, ampicillin, methicillin, cloxacillin and mitomycin C in small amounts/traces. Stained DNA could be feebly visualized directly in Sm run in agarose gel, which improved after its separation and concentration. These DNA materials were DNase sensitive, RNase and pronase resistant, and appeared to consist of fragments, c. less than or equal to 6 Mdal; this could repeatedly transform to SmR several recipient enterobacteria and vibrios; E. coli C600 and S. typhi 57, after such transformation revealed similar plasmid DNA bands that were absent in their wild-types. G + C mole% of plasmid and chromosomal DNA of recipient (57) along with that of Streptomyces griseus reference strain, suggested an extraneous origin for the plasmid DNA.

Anti-Bacterial Agents↗

PubMed1999

Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases.

Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Molecular genetic analysis of easily accessible breast tumour DNA, purified from tissue left over from hormone receptor measurement.

In order to establish a large panel of normal and tumour DNA from primary breast cancer patients, we looked for a source of easily accessible, good quality breast tumour DNA. Following routine hormone receptor analysis at the hospital the leftover pellets contained the nuclei from the tumour tissue. We collected 670 pellets over a period of 2 1/4 years and isolated a large amount of DNA (on average 400 microg per pellet). To control the quality of this tumour DNA, we analysed 41 pellets and matching normal DNA for loss of heterozygosity (LOH), with 11 microsatellite markers along chromosome 17. This chromosome is well described for breast cancer. LOH is a sensitive method, requiring good quality and pure tumour DNA. Contamination with normal DNA will blur the results. We found a high rate of LOH, ranging from 33 to 74%, which is in agreement with other reports, and therefore recommend this rich source of breast tumour DNA for molecular biological analysis.

Breast Neoplasms↗

PubMed1979

[Absence of anti double stranded DNA antibodies in viral hepatitis (author's transl)].

The presence of anti double stranded DNA antibodies (ds DNA Ab) in the sera of patients with acute viral hepatitis and B virus chronic active hepatitis has recently been described. In contrast with these results we found no ds DNA ab, but anti single stranded DNA antibodies (ss DNA ab) in the sera from patients with these diseases. The ds DNA ab were studied simultaneously by two methods: Immunofluorescence (IF) on crithidia luciliae and Farr test. In this later case it was shown that the ds DNA preparation contained no ss DNA because it did not react with an anti ss DNA rabbit serum. Our results indicate that the above mentioned authors have been using in their Farr test a preparation of ds DNA contaminated by ss DNA, thus revealing the presence of anti ss DNA ab but not anti ds DNA ab, in the sera of their patients. On the other hand the presence of ds DNA ab has been shown in the sera of 85% of the patients with systemic lupus erythematosus and of 20,5% of the patients with non EB chronic active hepatitis. At least one part of these later are probably autoimmune diseases.

Acute Disease↗