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PubMed1978

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength.

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.

Animals↗
PubMed2002

Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe.

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.

Bacteria, Anaerobic↗
PubMed1985

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization.

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.

Base Sequence↗
PubMed1986

Purification and characterization of Toxoplasma gondii tachyzoite DNA.

Toxoplasma gondii DNA from RH strain tachyzoites was purified from contaminating murine DNA by isopycnic density gradient ultracentrifugation in CsCl containing the fluorescent dye Hoechst 33258. Quantitative hybridization studies with DNA from uninfected mice and DNA extracted from oocysts of the ME49 strain of T. gondii confirmed that RH strain DNA could be obtained at least 90% pure with a high recovery. Preliminary characterization studies on such purified DNA showed that it had a G + C content of 50% and was not significantly methylated.

Animals↗
PubMed1981

Origin and characterization of short DNA chains in Escherichia coli.

One of the major problems in the study of DNA replications involves the presence of numerous short, non-nascent DNA chains in the cell. Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult. This complication can be avoided by the use of hydroxyapatite chromatography. Previous results from this laboratory had shown that short nascent DNA chains could be eluted from hydroxyapatite at low phosphate concentrations because of their non-covalent association with protein. Additional data now indicate that this isolation procedure yields a DNA preparation which contains only nascent DNA and is essentially free of non-nascent chains. 5'-Terminal labelling patterns and molecular weight distributions show that only nascent DNA chains from the DNA-protein complex and can therefore be separated from non-nascent chains if the isolation is performed under non-denaturing conditions. The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA-protein complex. Using this isolation technique, the 5'-terminal nucleotides of nascent DNA chains were determined in an effort to learn something about the chain initiation process. DNA was pulse-labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5' end with [32P]phosphate. Two-dimensional thin-layer chromatography of the 32P-labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5' end in approximately equal amounts. In most cases, this 5'-terminal nucleotide distribution does not vary significantly, even when the conditions of pulse-labeling are changed. Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5'-end nucleotides detected. In this case, the amount of dTMP at the 5' end decreases significantly, reflecting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides. From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process.

Chemical Phenomena↗
PubMed2026

Short-read genome skimming enables molecular barcoding of old myxomycete collections.

This study evaluates the effectiveness of Illumina-based genome skimming for barcoding myxomycete herbarium collections ranging from 29 to 91 years in age. We successfully retrieved partial sequences of the standard marker gene (nucSSU) in all cases, as well as additional markers (mtSSU, EF1a, and COI) for certain collections. Altogether, 28 genes were recognized in the studied material. In a 33-year-old specimen of Lindbladia tubulina, the assembly reached an N50 of 4.19 kb, enabling the recovery of extended functional loci. The input genomic DNA quantity emerges as the primary determinant of sequencing success. Samples with high DNA yields provide representative amounts of contigs coming confirmedly (matching sequences in the NCBI nucleotide database) or potentially (no-hit fraction) from myxomycetes, regardless of specimen age. In addition to target DNA, we revealed distinct signals of both anthropogenic contamination (human DNA and skin microflora) and natural substrate inhabitants, including oribatid mites and bacteria from dead wood, soil, and grass litter. Thus, even in old collections, metagenomic data still carry information regarding the substrate upon which the myxomycete developed. The results demonstrate that short-read genome skimming may help to integrate historical type material of myxomycetes into contemporary phylogenetic research. This method overcomes the length-dependent limitations of traditional Sanger sequencing, thus providing a roadmap for the future of museomics in myxomycetology.

Amoebozoa↗
PubMed2004

An unexpected experimental pitfall in the molecular diagnosis of bacterial endophthalmitis.

General primer-mediated ribosomal DNA amplification during endophthalmitis may improve the quality of diagnostic microbiology. However, extreme care needs to be taken not to introduce contaminating bacterial DNA during surgery procedures. The use of decontaminating iodine solutions can lead to such contamination due to the presence of DNA from Pseudomonas-like organisms.

Anti-Infective Agents, Local↗
PubMed2000

Ancient DNA analysis of human populations.

The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800-1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0-100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA.

Animals↗
PubMed2004

Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides.

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.

Animals↗
PubMed1972

Replication of mitochondrial DNA in mouse L cells and their thymidine kinase - derivatives: displacement replication on a covalently-closed circular template.

Thymidine kinase(-) lines of mouse L cells incorporate thymidine exclusively into mitochondrial DNA. This fact permits assessment of labeled mitochondrial DNA components in ethidium bromide-cesium chloride gradients. Contaminating nuclear DNA is unlabeled and need not be removed. Elimination of the DNase treatment of purified mitochondria reveals that the replicative forms that exhibit displacement replication up to full genome size are covalently-closed circular molecules. Denaturation followed by brief renaturation of these larger replicating molecules produces closed-circular DNA with a deficiency of Watson-Crick turns, appearing as single-strand loops. This result suggests that displacement replication proceeds with nicking and rapid closure of the covalently-closed circular template.

Animals↗
PubMed1976

A double antibody solid phase assay for DNA autoantibodies for clinical use.

DNA antoantibodies in serum will bind to antigen-coated polystyrene tubes and can be detected by radiolabelled anti-immunoglobulin. The method is quantitative, gives information on the antibody class and is not readily subject to interference by factors such as the size of the DNA, minor contamination of double-stranded with single-stranded DNA and the presence of materials which can combine "non-specifically" with the antigen. Double-stranded DNA gave good discrimination between SLE and rheumatoid arthritis but with single-stranded preparations approximately 40% of the RA patients showed elevated antibody values. Individual results obtained with the two antigens were compared and correlated with the Farr test and ANA titres. Surprisingly, a proportion of the SLE sera gave high background binding to tubes coated with gelatine.

Antibodies, Antinuclear↗
PubMedDate not supplied

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture.

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.

Adsorption↗
PubMed2002

Parvovirus B19 transmission by heat-treated clotting factor concentrates.

BACKGROUND: Human parvovirus B19 (B19) DNA can be frequently detected in plasma-derived coagulation factor concentrates. The production of some clotting factor products includes heat treatment steps for virus inactivation, but the effectiveness of such steps for B19 inactivation is unclear. Moreover, detailed transmission case reports including DNA sequence analysis and quantification of B19 DNA from contaminated heat-treated blood components have not been provided so far. Therefore, the correlation between B19 DNA in blood components and infectivity remains unclear. STUDY DESIGN AND METHODS: Asymptomatic B19 infections of two patients with hemophilia A were detected by anti-B19 seroconversion after administration of B19-contaminated heat-treated clotting factors. The suitability of nucleic acid sequence analysis for confirmation of B19 transmission was investigated. Furthermore, the B19 DNA level in blood components was determined and the drug administration was reviewed to calculate the amount of inoculated B19 DNA. RESULTS: Both B19 transmissions from clotting factor products could be confirmed by identical nucleic acid sequences of virus DNA from patients and blood components while sequences from unrelated controls could be differentiated. One patient received, for 4 days, a total of 180 mL vapor heat-treated prothrombin complex concentrate containing 8.6 x 10(6) genome equivalents per mL of B19 DNA. The other patient received 966 mL of low-contamination (4.0 x 10(3) genome equivalents/mL) dry heat-treated FVIII concentrate over a period of 52 days. CONCLUSION: B19 transmissions can be confirmed by nucleic acid sequencing. However, due to the low variability of the B19 genome, a large part of the B19 genome must be analyzed. The transmissions show that the applied heat treatment procedures were not sufficient to inactivate B19 completely.

Child, Preschool↗
PubMed2000

Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

Base Sequence↗