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PubMed1978

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

DNA, Bacterial↗
PubMed1975

Anatomy of herpes simplex virus DNA: strain differences and heterogeneity in the locations of restriction endonuclease cleavage sites.

Digestion of herpes simplex virus DNA by the HinIII or Eco RI restriction endonucleases yielded 11 to 15 fragments with molecular weights between 1 x 10(6) and 28 x10(6). The electrophoretic profiles obtained in 0.3% agarose gels with DNA fragments from none different strains of herpes simplex virus type 1 could be readily differentiated from the patterns exhibited by the corresponding fragments from four separate strains of type 2 virus; however, with each serotype, the laboratory strains differed significantly among themselves and also from isolates passaged a minimum number of times outside the human host. Digestion of all DNAs of herpes simples virus with either enzyme reproducibly generated two classes of fragments (major and minor) which differed in molar ocncentration. Moreover, although the molecular weight of an intact herpes simplex 1(F1) DNA molecule is approximately 98 x 10(6), the summed molecular weights of all major and minor HinIII fragments totalled 160 x 10(6), and the seven major fragments alone accounted for only 60 x 10(6). These unusual features indicate the existence of limited heterogeneity in the positions of cleavage sitet along individual molecules. We have eliminated the possibility that minor fragments arose from contamination with the defective DNA of high byoyant density which appears on serial undiluted passage of the virus. In fact, this latter type of DNA was resistant to cleavage by HinIII and gave large amounts of only two species of EcoRI fragments; suggesting that the defective molecules consist of many tandem repeats of a small segment of viral DNA. The heterogeneity in the viral DNA of normal density appears to be related to the structural organization of the molecules and does not necessarily imply differences in genetic content.

Carcinoma, Squamous Cell↗
PubMed1998

Contamination-free and automated composition of a reaction mixture for nucleic acid amplification using a capillary electrophoresis apparatus.

The acceptance of the polymerase chain reaction (PCR) as an amplification method in molecular diagnostics and the rapid development of capillary electrophoresis (CE) as an analysis method of those PCR products was a reason for us to investigate further integration of those two techniques. Using a fused-silica capillary as a pipette we were able to compose a PCR mixture in the CE apparatus. Because a capillary can be thoroughly rinsed and the CE apparatus is a closed system, the risk of contamination and therefore the occurrence of false positive results is minimized. The fact that a CE system can be fully automated contributes to a more reproducible and standardized PCR composition protocol.

Autoanalysis↗
PubMed2005

Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic.

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-microm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.

Arctic Regions↗
PubMed2004

An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants.

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.

Animals↗
PubMed1988

Enumeration of virulent Yersinia enterocolitica colonies by DNA colony hybridization using alkaline treatment and paper filters.

The efficiency of enumerating virulent Yersinia enterocolitica colonies was determined for 10 artificially contaminated foods by autoradiography after DNA colony hybridization using a 32P-labelled genetic probe and paper filters. Enumeration efficiency, which ranged from 0-98% (average 25 +/- 31% standard deviation), improved (42-138%, average 83 +/- 27% standard deviation) after enrichment with 0.5% KOH-0.5% NaCl. These efficiencies are equivalent to those obtained with nitrocellulose filters.

Autoradiography↗
PubMed1995

Competitor internal standards for quantitative detection of mycoplasma DNA.

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

Base Sequence↗
PubMedDate not supplied

Sample suitability for the detection of minor white cell populations (microchimerism) by polymerase chain reaction.

BACKGROUND: There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing. STUDY DESIGN AND METHODS: Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood. RESULTS: Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed. CONCLUSION: Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.

Anemia, Sickle Cell↗
PubMed1989

Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe.

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.

Animals↗
PubMed1989

Evaluation of deoxyribonucleic acid (DNA) isolated from human bloodstains exposed to ultraviolet light, heat, humidity, and soil contamination.

This study was designed to analyze the effects of common environmental insults on the ability to obtain deoxyribonucleic acid (DNA) restriction fragment-length polymorphisms (RFLP) patterns from laboratory prepared specimens. The environmental conditions studied include the exposure of dried bloodstains to varying amounts of relative humidity (0, 33, 67, and 98%), heat (37 degrees C), and ultraviolet light for periods of up to five days. In addition, the effect of drying over a four-day period in whole blood collected with and without ethylenediaminetetraacetate (EDTA) was examined. The results of the study showed that, under the conditions studied, the integrity of DNA is not altered such that false RFLP patterns are obtained. The only effect observed was that the overall RFLP pattern becomes weaker, but individual RFLP fragments are neither created nor destroyed.

Blood Stains↗
PubMed1999

Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography.

We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feed-stock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography. The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.

Anion Exchange Resins↗
PubMed1997

Parallel overlap assembly for the construction of computational DNA libraries.

Algorithms for computing with DNA currently require the construction of pools of molecules in which each distinct molecule represents a different starting point for the calculation. We have begun building such pools using the technique of parallel overlap assembly that is already used for the generation of diversity in biologically useful combinatorial search techniques such as gene shuffling. Unlike these applications, a pool in a molecular computer must be complete, containing all possible strands, and ordered, having minimal contamination from incorrectly assembled DNA. We present an experiment in which parallel overlap assembly is used to construct a computational pool and an experiment in which this pool is used to solve the NP-complete maximal-clique problem.

Algorithms↗
PubMed2005

Mosaics of ancient mitochondrial DNA: positive indicators of nonauthenticity.

Research into ancient mitochondrial DNA is plagued by contamination, post mortem damage, and other artefacts. The stringent set of controls suggested by Cooper and Poinar a few years ago are, however, rarely followed in practice, and even when applied carefully, these criteria need not be sufficient to guarantee authenticity. The fairly relaxed prerequisites now common for ancient population studies have opened the door for all kinds of contamination and sequencing errors to enter ancient mtDNA data. To reject or question authenticity of particular sequencing results a posteriori, one can follow similar strategies of focused database comparisons that have proven to be effective and successful in the case of flawed modern mtDNA data.

Artifacts↗
PubMed2005

Analysis of oxidative DNA damage 8-hydroxy-2'-deoxyguanosine as a biomarker of exposures to persistent pollutants for marine mammals.

An oxidative DNA biomarker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was determined in the livers and kidneys of stranded or by-caught cetaceans along the Taiwan coast through isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to evaluate the feasibility of analyzing the DNA adduct in marine mammals and then to study the association between 8-OHdG and levels of polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) in the blubbers of the cetaceans. The mean values of the 8-OHdG from the liver and kidney samples were 19.83+/-10.00 pmol/micromol deoxyguanosine (dG) (6.90-53.53 pmol/micromol dG) and 19.16+/-7.48 pmol/micromol dG (5.36-39.36 pmol/micromol dG), respectively. In general, 8-OHdG was not related to the general health status of the by-caught and stranded animals and also was not related to species. However, the levels of 8-OHdG had a positive correlation with concentrations of PCBs, but not DDE, in female cetacean livers. In addition, when selected coplanar PCBs (dioxin-like congeners) were used to compare the 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) with 8-OHdG of by-caught cetaceans, a high positive correlation (r = 0.80, p < 0.01) was found in mature female animals. Thus, the detection of 8-OHdG in marine mammals with isotope-dilution LC/MS/ MS is possible, and the study of the relationship between oxidative DNA damage and environmental contaminants under natural exposure indicates that the level of 8-OHdG in female cetacean livers is associated with coplanar PCBs and the factor of sexual maturity.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1987

[Detection and identification of Mycoplasma infections by DNA hybridization].

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.

Animals↗
PubMed1995

Simultaneous DNA 'fingerprinting', diagnosis of sex and single-gene defect status from single cells.

Sex and cystic fibrosis status have been previously diagnosed separately at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosis of sex, cystic fibrosis and a DNA 'fingerprint' within a single reaction from a variety of single cells. As contamination cannot be totally excluded, particularly at the single cell level, DNA 'fingerprinting' can be used to assess the risk of contamination. High sensitivity with single cells is combined with very high specificity (estimated matching probability of 10(-7)-10(-8)), allowing the source of the amplified cell to be identified with a very high degree of probability. Fluorescent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was used to diagnose sex, and primers for the CFTR region were used to determine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan software. This technique has many applications such as prenatal and preimplantation diagnosis, forensic identification of small or degraded samples, and detection of contamination sources. DNA fingerprints of single haploid spermatozoa and other cells can be assessed, so ensuring the detection of both diploid and haploid contamination during preimplantation diagnosis.

Cystic Fibrosis↗
PubMed1989

Detection of Epstein-Barr virus DNA by polymerase chain reaction in blood and tissue biopsies from patients with Sjogren's syndrome.

Polymerase chain reaction has been used to detect increased levels of EBV DNA in salivary gland (SG) biopsies and PBL from patients with Sjogren's syndrome (SS). These results suggest that EBV, which has a normal site of latency in a small number of SG epithelial cells, may be reactivated in SS patients and provide a target for immune attack. The great sensitivity of polymerase chain reaction (PCR) and the ability to analyze very small tissue biopsies (37) make this technique well suited for clinical diagnosis. Specific methods to prevent artefactual contamination of tissue biopsy DNA with viral DNA of other samples (i.e., lyophilization of samples before DNA extraction) and the use of an internal positive control (i.e., inclusion of primers for a single copy human gene) during PCR amplification are presented. Since EBV reactivation occurs with markedly increased frequency in patients with lymphoproliferative and immunodeficiency diseases, as well as transplant recipients receiving cyclosporin A (10), rapid methods of viral detection such as PCR may allow better monitoring of medications and early detection of EBV-related lymphomas that may arise in these patients.

Base Sequence↗