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PubMedDate not supplied

Radical-induced DNA damage by a synthetic analogue of copper(II)-bleomycin.

The capacity of free-radical formation and concomitant radical-induced DNA damage by [Cu(PMA)]X (X = ClO4-, BF4-), the first crystalline synthetic analogue of Cu(II)-BLM, have been studied. In phosphate buffer, [Cu(PMA)]+ can be reduced by thiols like DTT to a Cu(I) species that can be reoxidized with dioxygen. During oxygenation of the Cu(I) analogue, appreciable amounts of .OH radical are formed. This fact has been established by spin-trapping experiments. Effects of SOD and catalase as well as pH on the ESR signal of the spin adduct have identified a set of reactions that eventually leads to .OH radical formation by [Cu(PMA)]+. Interestingly, the same reaction scheme has been proposed earlier for Cu(II)-BLM. In the presence of DTT and dioxygen, [Cu(PMA)]+ inflicts significant damage to plasmid DNA. That the damage does not arise from iron contamination has been established. The DNA damage is hindered when SOD or catalase is present in the incubation mixture. Less strand scission is observed at higher pH. Modulation of DNA cleavage efficiency with systematic variations in DTT concentration indicates that the conflicting reports on the ability of Cu(II)-BLM to mediate in vitro DNA damage might have resulted from the different DTT concentrations used by different groups.

Bleomycin↗
PubMed1999

Combination of biomolecular and stable isotope techniques to determine the origin of organic matter used by bacterial communities: application to sediment.

Natural isotopic composition is a good tool to trace organic matter in ecosystems. Recent studies used a combination of molecular and stable isotope techniques to determine the origin of the organic carbon used by bacteria in the water column. In our study, we show that this procedure can be used for analysis of sediment bacterial communities with few modifications. In the water column, bacterial recovery is done before DNA extraction. In the sediment, we tested qualitatively and quantitatively a direct and indirect extraction of DNA. The direct extraction was the most efficient. It recovered between 3.1 and 15.8 microg DNA g(-1) dry sediment and the contamination of field samples by eucaryotic DNA was less than 13%. In this preliminary study of the salt marsh ecosystem, the delta(13)C values of DNA (-26 to - 24%) recovered from the sediment were close to the delta(13)C values of halophytic plants (-26.4 and - 25.3%) showing a relationship between plants and microorganisms. Thus, this procedure can be used to trace the flow of carbon through the sediment microbial biomass and to understand the variation of bacterial activity according to the inputs of allocthonous and autochtonous organic matter.

Bacteria↗
PubMed2004

Quantitative real-time detection of parvovirus B19 DNA in plasma.

BACKGROUND: As of 2004, the European Pharmacopoeia demands that plasma pools for production of anti-D immunoglobulin should not contain more than 104 IU per mL of parvovirus B19 (B19V) DNA. Hence, before pooling, highly viremic donations have to be identified, and after pooling the level of B19V DNA must be determined. The performance of a new real-time B19V DNA PCR test (Roche, Mannheim, Germany) was studied, using a DNA extractor (NucliSens, bioMerieux, Boxtel, the Netherlands) for isolation of nucleic acid, and using a DNA quantification test (LightCycler apparatus, Roche, Mannheim, Germany) for amplification and detection. STUDY DESIGN AND METHODS: Dilutions of the international B19V DNA standard and reference preparations were tested to determine the precision, linear range, and accuracy of the assay and to calculate the factor for conversion of B19V DNA copies to IUs. The internal control signals, invalid test results, and the effect of cryo-poor plasma were studied as a measure for robustness. Routine performance was assessed by testing 164 manufacturing pools (not screened for B19V) and 1048 test pools of 480 donations each. RESULTS: The copies-to-IU conversion factor was calculated to be 3.34 (95% CI, 3.07-3.63). The assay appears linear between 10(3) and 10(7) IU per mL. Between 10(3) and 10(5) IU per mL, the test can discriminate samples differing a factor two in B19V DNA content. Overall, 0.78 percent of the test results were invalid. Of 127 B19V DNA negative control plasma samples, 7 were contaminated with low levels of B19V DNA. Of 164 nonscreened manufacturing plasma pools, 92 contained B19V DNA (56%); 13 contained more than 10(4) IU per mL. Of 503,040 donations, 29 contained more than 5 x 10(6) IU per mL B19V DNA (1:17,346). CONCLUSION: The B19V DNA quantification test (LightCycler, Roche ) is suitable for quantitative, routine, in-process measurement of B19V DNA levels in plasma pools, using the DNA extractor (NucliSens, bioMerieux) for nucleic acid isolation.

Blood Donors↗
PubMed1977

Sequence arrangement in satellite DNA from the muskmelon.

Two fractions of a satellite DNA from the muskmelon (Cucumis melo L.) isolated as a unimodal peak from CsCl gradients, differ in melting properties and complexity as estimated by reassociation kinetics. At 49.8 C, all of the low melting fraction was denatured and all of the high melting fraction was native. There were almost no partially denatured molecules detected in the electron microscope at this temperature. This observation provides direct evidence that the two fractions are not closely linked. We conclude that satellite I, the high t(m), low complexity fraction, exists as a 600-nucleotide sequence in blocks of at least 67 tandem repeats. Since the complexity of the low melting fraction, satellite II, is greater than the size of the molecules in our assay, we can only say that the minimum size of each unit of satellite II is 2.5 x 10(7) daltons. It is unlikely that any spacer sequences are interspersed with either satellite.Sequences homologous to those of satellite I were also shown to be present as a minor fraction on 4900 nucleotide pair fragments with main band DNA density. These long main band fragments probably contain in addition at least two repeated sequence elements unrelated to satellite I since they aggregate (form large network structures) when reassociated. Coaggregation of sheared (3)H-satellite I with long main band DNA could not be attributed to contamination of main band with long satellite DNA. We interpret the results as an observation of a recently created family of tandemly repeating sequences whose members are beginning to be scattered throughout the genome.We discuss how the aggregation technique may be generally useful for assessing linkage between a minor and a major DNA fraction when both fractions may be present in the initial DNA preparation. Applications for the technique include the search for DNA sequences in the nucleus which are homologous with chloroplast DNA and for Agrobacterium tumefaciens DNA in the nuclei of plant cells transformed to the tumor phenotype by the bacterium.

Journal Article↗
PubMed2006

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Automation↗
PubMed1999

A systematic analysis of PCR contamination.

In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.

DNA↗
PubMed2026

Cleanifier: contamination removal from microbial sequences using spaced seeds of a human pangenome index.

MOTIVATION: The first step when working with DNA data of human-derived microbiomes is to remove human contamination for two reasons. First, many countries have strict privacy and data protection guidelines for human sequence data, so microbiome data containing partly human data cannot be easily further processed or published. Second, human contamination may cause problems in downstream analysis, such as metagenomic binning or genome assembly. For large-scale metagenomics projects, fast and accurate removal of human contamination is therefore critical. RESULTS: We introduce Cleanifier, a fast and memory frugal alignment-free tool for detecting and removing human contamination based on gapped k-mers, or spaced seeds. Cleanifier uses a pangenome index of known human gapped k-mers, and the creation and use of alternative references is also possible. Reads are classified and filtered according to their gapped k-mer content. Cleanifier supports two filtering modes: one that queries all gapped k-mers and one that queries only a sample of them. A comparison of Cleanifier with other state-of-the-art tools shows that the sampling mode makes Cleanifier the fastest method with comparable accuracy. When using a probabilistic Cuckoo filter to store the complete k-mer set, Cleanifier has similar memory requirements to methods that use a sampled minimizer index. At the same time, Cleanifier is more flexible, because it can use different sampling methods on the same index. AVAILABILITY AND IMPLEMENTATION: Cleanifier is available via gitlab (https://gitlab.com/rahmannlab/cleanifier), PyPi (https://pypi.org/project/cleanifier/), and Bioconda (https://anaconda.org/bioconda/cleanifier). The pre-computed human pangenome index is available at Zenodo (https://doi.org/10.5281/zenodo.15639519).

Humans↗
PubMed2005

DNA damage evaluated by the comet assay in lymphocytes of children with 137Cs internal contamination caused by the Chernobyl accident.

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable 137Cs internal contamination caused by food consumption. The children were selected for the study immediately after a 137Cs whole body counter measurement of internal contamination. The minimal detectable amount of 137Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 microSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 +/- 5.7 AU) versus exposed (8.5 +/- 4.8 AU) groups were found. These results suggest that low doses of 137Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.

Adolescent↗
PubMed2000

Bacteria of asymptomatic periradicular endodontic lesions identified by DNA-DNA hybridization.

Possible inclusion of contaminant bacteria during surgery has been problematic in studies of periradicular lesions of endodontic origin. Therefore, in this study, two different surgical techniques were compared. A second problem is that some difficult to cultivate species may not be detected using bacteriological methods. Molecular techniques may resolve this problem. DNA-DNA hybridization technology has the additional advantage that DNA is not amplified. The purpose of this investigation was to determine if bacteria from periradicular endodontic lesions could be identified using DNA-DNA hybridization. A full thickness intrasulcular mucoperiosteal (IS) flap (n = 20) or a submarginal (SM) flap (n = 16) was reflected in patients with asymptomatic apical periodontitis. DNA was extracted and incubated with 40 digoxigenin-labeled whole genomic probes. Bacterial DNA was detected in all 36 lesions. Seven probes were negative for all lesions. In patients with sinus tract communication, in teeth lacking intact full coverage crowns, and in patients with a history of trauma 4-13 probes provided positive signals. Seven probes were positive in lesions obtained by the IS, but not the SM technique. Two probes were in samples obtained with the SM technique, but not the IS. Only Bacteroides forsythus and Actinomyces naeslundii genospecies 2 were present in large numbers using either the IS or the SM technique. The SM flap technique, in combination with DNA-DNA hybridization, appeared to provide excellent data pertaining to periradicular bacteria. These results supported other studies that provide evidence of a bacterial presence and persistence in periradicular lesions.

Actinomyces↗
PubMed2005

Cytochrome P4501A induction and DNA adduct formation in glaucous gulls (Larus hyperboreus), fed with environmentally contaminated gull eggs.

This study indicates that complex mixtures of pollutants found in the Arctic marine environment have genotoxic effects in glaucous gulls (Larus hyperboreus). DNA adducts were quantified, by the (32)P-postlabeling technique, in liver samples from gulls fed with hen eggs (controls) and from gulls fed with environmentally contaminated gull eggs (exposed). All birds were grown and fed under laboratory conditions. Hepatic homologues to mammalian cytochrome P4501A (CYP1A) proteins were also determined by Western blotting. DNA adducts were detected in all but one liver sample, but the exposed birds had a significantly increased level of DNA adducts relative to that of the controls. There was no clear significant correlation between the DNA adduct level and the level of organochlorine compounds (OCs) in blood. The level of CYP1A protein was significantly higher in the liver of exposed male gulls than in the liver of control males and positively correlated, with significance, to the level of OC compounds measured in blood. There was no significant correlation between the level of DNA adducts and the CYP1A protein content.

Animals↗
PubMed2003

DNA analysis of neonatal human remains wrapped and kept in a vinyl bag for 15 years.

DNA analysis of a newborn baby wrapped and kept in a vinyl bag for 15 years was performed. DNA isolated from the femur and humerus was used to determine the sex and kinship between the infant and the putative parents. Amplification of mtDNA, ABO, HLA, CST3, CST5, VWA, D12S66, D21S11, CSF1PO, TPOX, THO1 and 10Y polymorphisms and the amelogenin gene was carried out. Several mtDNA types were obtained, suggesting that the sample was contaminated by exogenous DNAs. One of the DNA samples obtained from the femur showed an identical mtDNA sequence to that of the mother except for one site, and this pattern was also found in another DNA sample. None of our laboratory personnel had that type, so we thought it was possible that this sample contained the target DNA. However, maternity was denied by the CST3 polymorphism. Finally, we concluded that the sample had been contaminated with exogenous DNA before we started to examine the body. Although it is difficult to determine the sources of this contamination, PCR amplification from highly degraded DNA is very sensitive to such contamination, and we must be even more careful in DNA analysis of such samples than in that of not so severely degraded specimens.

DNA↗
PubMed1995

Origin of the foraminifera.

We report nuclear small-subunit rDNA sequences for two species of the benthic foraminifer Ammonia. Because of their abundance in present and past seas, the foraminifera are a very important group of organisms used in a wide range of geologic studies; however, the ancestry of this group was, until now, unknown. Difficulties in purifying foraminiferal DNA and avoiding PCR contamination led us to develop DNA.DNA in situ hybridization techniques to verify our sequence data. Phylogenetic analysis of verified foraminiferal DNA sequence indicates these taxa are a divergent, "alveolate" lineage, within the major eukaryotic radiation. Our findings cast doubt upon the assumption that all foraminifera are derived from an amoebalike ancestor. Cytological features and the DNA sequences presented herein suggest that the foraminifera were derived from a heterokaryotic flagellated marine protist, probably sometime in the later Proterozoic.

Animals↗
PubMed1994

Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase.

Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA.

Animals↗
PubMedDate not supplied

[Biochemical heterogeneity of mitochondria].

Rat liver mitochondria were fractionated on the basis of their sedimentation coefficients in the gradient of ficoll. The fractions ("heavy", "middle" and "light" mitochondria) were heterogeneous with regard to the content of protein, DNA, cytochrome a + a3 and respiratory activity. Heterogeneity of mitochondria did not result from the damage or microsomal and lysosomal contamination. The biosynthesis of DNA, RNA and proteins in the different fractions of mitochondria was studied. In vivo incorporation of radioactive precursor into RNA was highest in the fractions of "middle" mitochondria, whereas in vitro the "heavy" mitochondria showed maximum activity in the synthesis of RNA. In vitro DNA synthes was maximum in the fractions of "heavy" mitochondria, protein synthesis in "heavy" and "light" mitochondria. Activity of the synthesis of RNA, DNA and proteins in vitro depends on the content of DNA and cytochrome a + a3 in the different fractions of mitochondria. It is supposed that heterogeneity of mitochondria may be connected with their biogenesis.

Animals↗
PubMed2003

Evaluation of F+ RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters.

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.

Animals↗
PubMed1990

The effect of various stain carriers on the quality and quantity of DNA extracted from dried bloodstains.

Bloodstains were made with 200 microliters blood on each of 11 different common substrates to examine the effect of the stain carrier on the amount and quality of DNA recoverable. High-molecular-weight DNA was extracted from all samples after 2 days. The yield of DNA from each sample varied considerably, not only between the different stain carriers but also within a given category. With a DNA yield of up to 10 micrograms, paper, glass, nylon, wood, smooth leather and wool gave the best results, followed by blue denim and wallpaper (up to 6 micrograms), cotton fabric and carpeting (up to 4 micrograms) and suede (up to 2 micrograms). For several stain carriers the DNA-containing solution was contaminated by chemical substances, which in the case of the blue denim, suede, and carpet samples inhibited the digestion of the DNA with restriction enzymes and prevented DNA typing. The different textures of the stain carriers tested and (as for varying yields on the same carrier) the differing degree of loss of DNA during extraction and the physiological variation in the number of leukocytes in human blood are discussed as possible reasons for the wide range of variation in the amounts of DNA it was possible to extract.

Blood Stains↗
PubMed1990

Immunosuppressive effects of four trichothecene mycotoxins.

The trichothecenes are a family of mycotoxins contaminating some food and feeds. We have studied the inhibitory effect of four of them including less toxic compounds: T-2 toxin, DAS, DON and Trichodermin, on normal human peripheral blood lymphocytes and murine splenic lymphocytes. The results show that even for the least toxic compound, inhibiting concentrations can be realised in the blood by common alimentary contamination.

Animals↗