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At least 487 records · Page 27Linked to original sources
PubMedDate not supplied

An improved method for the isolation of supercoiled DNA molecules using ion-exchange column chromatography.

An improved procedure is presented for the rapid isolation of supercoiled molecules using ion-exchange chromatography of alkali-denatured material. Under the conditions employed, contaminating cellular DNA, RNA, and nicked circular molecules are denaturable and are presented to the column as single strands while intact, covalently closed, supercoiled molecules resist denaturation and are batch eluted from the column as the only remaining duplex species. Small amounts of protein and short, duplex fragments (less than 1 kb) are removed in the void volume. There is no need for gradient fractionation. Supercoil fractions are virtually free of contamination with linear molecules as determined by electron microscopy, have an A260 nm/A280 nm ratio approaching 2.0, and are suitable for subsequent heteroduplex analysis, molecular cloning, subcloning, or restriction enzyme mapping. The method has been used successfully to purify plasmids up to 45 kb in length and to isolate rare circular replication intermediates from an overwhelming excess of contaminating linear molecules in retrovirus-infected cells.

Chromatography, Ion Exchange↗
PubMed1998

Specific ribosomal DNA sequences from diverse environmental settings correlate with experimental contaminants.

Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella (formerly Zoogloea), Acinetobacter, Stenotrophomonas, Escherichia, Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.

Animals↗
PubMed2006

Allelic losses of chromosome 10 in glioma tissues detected by quantitative single-strand conformation polymorphism analysis.

BACKGROUND: Detection of loss of heterozygosity (LOH) in clinical tissue samples is frequently difficult because samples are usually contaminated with noncancerous cells or because tumor cells in single tissues have genetic heterogeneity, and the precision of available techniques is insufficient for reliable analysis in such materials. We hypothesized that single-strand conformation polymorphism (SSCP) analysis can precisely quantify the gene dosage in mixed samples and is suitable for detection of LOH in clinical tissue samples. METHODS: We assessed the accuracy of a fluorescent SSCP method for the quantification of single-nucleotide polymorphism (SNP) alleles, using DNAs that were composed of cancerous DNA mixed with noncancerous DNA at various ratios. We applied this method to precisely characterize LOH in glioma tissue samples, using 96 SNPs that were evenly distributed throughout chromosome 10. RESULTS: LOH could be detected even in the cancerous DNA heavily contaminated (up to 80%) with noncancerous DNA. Using this method, we obtained LOH profiles of 56 gliomas with resolution at the SNP level (i.e., 1.5-Mbp interval). Anaplastic astrocytomas exhibited both 10p and 10q LOH, whereas diffuse astrocytomas frequently (63% of the cases) exhibited loss of 10p alone. We also found a possible new LOH region (around 10p13) in gliomas. CONCLUSIONS: The present method is effective for precise mapping of LOH region in surgically obtained tumor tissues and could be applicable to the genetic diagnosis of cancers other than gliomas.

Alleles↗
PubMed2003

The fingernails of Mary Sullivan: developing reliable methods for selectively isolating endogenous and exogenous DNA from evidence.

The fingernails of Mary Sullivan, the last victim of the Boston Strangler, were examined to determine if any genetic information about the murderer could be obtained. The nails were extremely friable necessitating the development of new techniques for isolating and purifying DNA. DNA yields from nails were optimized by using a NaOH-based preparation technique, which was simple, efficient, and minimized handling. Methods for selectively and thoroughly removing exogenous material on nails were also developed through use of a species-specific PCR assay, wherein mitochondrial DNA from the nail could easily be differentiated from DNA of contaminating cells.

Animals↗
PubMed1988

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity.

In the present study, calf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5 while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.

Affinity Labels↗
PubMedDate not supplied

Visceral leishmaniasis in Teresina, n. e. Brazil: towards a DNA probe kit and its adaptation to processing blood-contaminated samples.

The Lmet2 chemiluminescent DNA probe is a valuable tool for identifying parasites of the Leishmania donovani -complex in sand flies, dogs and human samples. Recent blood meals in sand flies or blood contamination of tissue samples inhibited probe sensitivity, whether radiolabelled or chemiluminescent detection systems were used. Treatment of membranes with protease before hybridisation restored positive signal. Alternatively samples could be lysed with protease and applied to membranes with a vacuum blotting apparatus. The Lmet2 protocol provides the basis for a DNA probe kit that is adaptable for use with a wide range of other probes.

Animals↗
PubMed2003

Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations.

A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.

Chemical Precipitation↗
PubMed2002

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish, Cherax destructor Clark (Parastacidae: Decapoda).

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.

Animals↗
PubMed2003

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.

Cells, Cultured↗
PubMed2004

Affinity purification of plasmid DNA by temperature-triggered precipitation.

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.

Chemical Precipitation↗
PubMed2005

Relatively well preserved DNA is present in the crystal aggregates of fossil bones.

DNA from fossil human bones could provide invaluable information about population migrations, genetic relations between different groups and the spread of diseases. The use of ancient DNA from bones to study the genetics of past populations is, however, very often compromised by the altered and degraded state of preservation of the extracted material. The universally observed postmortem degradation, together with the real possibility of contamination with modern human DNA, makes the acquisition of reliable data, from humans in particular, very difficult. We demonstrate that relatively well preserved DNA is occluded within clusters of intergrown bone crystals that are resistant to disaggregation by the strong oxidant NaOCl. We obtained reproducible authentic sequences from both modern and ancient animal bones, including humans, from DNA extracts of crystal aggregates. The treatment with NaOCl also minimizes the possibility of modern DNA contamination. We thus demonstrate the presence of a privileged niche within fossil bone, which contains DNA in a better state of preservation than the DNA present in the total bone. This counterintuitive approach to extracting relatively well preserved DNA from bones significantly improves the chances of obtaining authentic ancient DNA sequences, especially from human bones.

Base Sequence↗
PubMed1995

Validation of mitochondrial DNA sequencing for forensic casework analysis.

Two sets of studies were performed to evaluate the forensic utility of sequencing human mitochondrial DNA (mtDNA) derived from various tissues and amplified by the polymerase chain reaction (PCR). Sequencing was performed on a Perkin-Elmer/Applied Biosystems Division (PE/ABD) automated DNA sequencer (model 373A). The first set of experiments included typical validation studies that had previously been conducted on forensic DNA markers, such as: chemical contaminant effects on DNA from blood and semen and the effect of typing DNA extracted from body fluid samples deposited on various substrates. A second set of experiments was performed strictly on human hair shafts. These studies included typing mtDNA from hairs that were: (1) from different body areas, (2) chemically treated, (3) from deceased individuals, and (4) deliberately contaminated with various body fluids. The data confirm that PCR-based mtDNA typing by direct automated sequencing is a valid and reliable means of forensic identification.

Animals↗
PubMed1993

Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection.

7-Methylguanine (7-meG) could be a useful marker of recent past exposure to environmental methylating agents for use in epidemiological studies. A method is described that is appropriate for such an application. 7-meG was released from DNA by thermal hydrolysis under conditions (pH 9, 70 degrees C, 8 h) that preferentially released the base from DNA rather than RNA and, following immunopurification using antibodies specific for this DNA adduct, quantification was achieved either by HPLC with electrochemical detection (ECD) or by ELISA. The detection limits of the two approaches were 0.5 and 2 pmol 7-meG/DNA sample respectively. 7-meG was analysed in DNA samples contaminated with known amounts of RNA to test the possible interference in the analysis by the minor modified nucleoside 7-methylguanine, which is present as a normal component of RNA. 7-meG levels measured in human pancreas and untreated rat liver DNA were between 2 and 7 pmol 7-meG/mumol guanosine and this level could not be explained by RNA contamination. The combination of immunoaffinity purification and HPLC with ECD provides a method that is sensitive and specific for 7-meG and suitable for integration into molecular epidemiological studies.

Animals↗
PubMed1992

A quick and inexpensive method for removing polysaccharides from plant genomic DNA.

A quick and inexpensive method has been demonstrated to remove polysaccharide contamination from plant DNA. Isolated plant genomic DNA with polysaccharide contaminants was dissolved in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) with NaCl ranging from 0.5-3.0 M, then precipitated with two volumes of ethanol. Most of the polysaccharides were removed effectively in a single high-salt precipitation at 1.0-2.5 M NaCl. At 3.0 M NaCl, the salt precipitated out of solution. Purified DNA was easily digested by either HindIII or EcoRI and was satisfactory as a template for PCR. The results show that high-salt precipitation effectively removed polysaccharides and their inhibitory effects on restriction enzyme and Taq polymerase activity.

DNA↗
PubMed2000

Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3' terminus to reduce false-positive signals.

A reverse transcription PCR (RT-PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3' terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 degrees C or 45 degrees C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 degrees C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.

3' Untranslated Regions↗
PubMed2000

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin.

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.

Apoptosis↗
PubMed1980

Presence and quantification of cell substrate DNA in inactivated poliovirus vaccine.

In order to follow the fate of cell substrate DNA in inactivated poliovirus vaccine (IPV), experiments simulating different steps in IPV preparation were performed. For this purpose, 3H-thymidine-labeled HeLa cell DNA released during lytic infection with Mahoney type 1 poliovirus strain was used as tracer. Low speed centrifugation (1500 xg for 15 min.) of crude virus suspension removed on the average 98.5% of the total labeled cell substrate DNA. Repeated freezing and thawing of the crude virus suspension before centrifugation did not increase the residual 1.5% radioactivity found in the supernatant. This level of contamination was not significantly influenced by filtration of the supernatant through a 0.22 micrometer pore size membrane filter. DEAE-Sepharose CL-6B chromatography of poliovirus suspensions containing large amounts of 3H-thymidine labeled HeLa cell extracts completely removed the contaminating substrate DNA. These data showed that IPV free of cell substrate DNA can easily be obtained by a usual purification procedure.

Centrifugation↗