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PubMed1979

Isolation and characterization of germ line DNA from mouse sperm.

Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas.

Animals↗
PubMed2006

Use of SEC-ICP-MS with a collision cell for determining the interaction of chromium with DNA extracted from metal-contaminated soils.

The potential of chromium to bind to DNA isolated directly from soil microbial communities was investigated in this study. An analytical scheme was developed to distinguish between chromium bound to DNA and its fragments or chromium contained elsewhere in an environmental DNA extract. DNA was extracted from chromium-contaminated soils and purified using DNA clean-up resins. Size-exclusion chromatography was employed due to its advantages in the separation and molecular weight approximation of large biomolecules. It was coupled with two on-line detection systems (spectrophotometric and inductively coupled plasma mass spectrometric) to study the binding of chromium to DNA or other components in a DNA extract. A collision cell was pressurized with helium to remove diatomic and polyatomic interferents resulting from the chosen mobile phase. Chromium peaks were observed in both the large and small molecular weight regions of the chromatogram; to further confirm that the environmentally extracted DNA contained Cr, the subsequently purified DNA was examined for total Cr using flow injection ICP-MS to accommodate small sample volumes. DNA samples isolated from the two soils examined contained 0.5-0.7 ppb Cr, indicating that DNA isolated directly from a chromium-contaminated soil has chromium bound to the nucleic acids.

Chromatography, Gel↗
PubMed1988

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.

Buffers↗
PubMed1996

Development of a multiplex PCR assay for the simultaneous detection and discrimination of HIV-1, HIV-2, HTLV-I and HTLV-II.

BACKGROUND: Multiplex polymerase chain reaction (PCR) has been established as a general technique for the simultaneous amplification of different target sequences. Uses of multiplex include pathogens identification, linkage analysis and genetic disease diagnosis. The high sensitivity of PCR may produce false-positive results due to contamination with previously amplified material. OBJECTIVES: To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 (HIV-1/2) and human T-lymphotropic virus types 1 and 2 (HTLV-I/II) proviral sequences. Such a method should incorporate a system that prevents the occurrence of false-positive results. STUDY DESIGN: Combinations of four primer pairs, one for each retrovirus, were assayed in order to determine the combination of oligonucleotides as well as the PCR conditions that yield the most specific and sensitive coamplification of proviral sequences. To prevent contamination with DNA from previous PCR amplifications, the uracil N-glycosylase (UNG) system was incorporated into the coamplification format. RESULTS: A combination of primer pairs from the gag region of HIV-1, env of HIV-2, pol of HTLV-I and tax of HTLV-II yielded specific and sensitive coamplification of proviral sequences. The UNG system was incorporated and shown to be efficient in the degradation of contaminating DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected 20/22 HIV-1, 8/10 HIV-2, 8/8 HTLV-I and 8/8 HTLV-II infections.

Cell Line↗
PubMed1994

PCR and reverse dot hybridization for the detection of endogenous retroviral transcripts.

Two degenerated oligonucleotide primers, known to amplify a fragment of the pol gene in all retroviruses tested so far have been used to amplify pol related sequences from human genomic DNA. Cloning and sequencing these fragments confirm a retroviral relationship for most of them and define 96 groups on the basis of their internal similarity. 96 pol fragments were probed with PCR amplified cDNA in reverse dot hybridization to investigate pol related transcripts. PCR amplified genomic DNA served as a control for contamination of genomic DNA in the RNA preparations. Isopycnic centrifugation in cesium trifluoroacetate yielded RNA with the lowest possible amounts of contaminating DNA. This technique is a powerful and a well-controlled tool for the detection of endogenous retroviral transcripts and may be helpful for investigating the involvement of endogenous retroviruses in various diseases.

Amino Acid Sequence↗
PubMed1993

Mutational analysis using denaturing gradient gel electrophoresis and PCR.

Denaturing gradient gel electrophoresis (DGGE) separates (DNA) molecules based on their sequence. Using the proper conditions, all base-pair substitutions can be resolved from the wild-type sequence using DGGE. Polymerase chain reaction (PCR) permits rapid amplification of a given region of the genome. In this paper, we demonstrate the utility of DGGE combined with PCR for mutation analysis by presenting different examples: (i) analysis of mouse p53 cDNA for mutations, (ii) simultaneous analysis of thousands of 4NQO-induced mutants for mutations in HPRT exon 3, (iii) examination of the fidelity of the thermostable DNA polymerase isolated from Pyrococcus furiosus (Pfu), (iv) purification of mutant DNA from contaminating wild-type DNA from mouse spleenic T-cell clones.

4-Nitroquinoline-1-oxide↗
PubMed1994

Analysis of in vivo mutation in exon 8 of the rat hprt gene.

We have analyzed mutations in exon 8 of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in T-lymphocytes from the spleens of ethylnitrosourea-treated female rats. Presumptive hprt- mutants were isolated by clonal growth in the presence of 6-thioguanine. DNA from 6-thioguanine-resistant colonies was amplified by the polymerase chain reaction using intronic primers flanking hprt exon 8. The identification of mutant sequences and the separation of mutant DNA from contaminating wild-type DNA was accomplished by denaturing gradient gel electrophoresis. Of 118 clones analyzed, 19 contained mutations and DNA sequence analysis identified eight unique sequence alterations. We also used single-strand conformation polymorphism analysis to screen for mutations in the same fragment of the hprt gene. This analysis was less successful than denaturing gradient gel electrophoresis in detecting the eight unique mutations. The procedures described here may represent a useful approach for studying the mechanisms of in vivo mutation.

Animals↗
PubMed1999

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

Alleles↗
PubMed2004

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations.

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

Biomedical Engineering↗
PubMed1993

[Detection of HBV DNA in human tears and its contamination of the tonometer].

HBV DNA was detected by PCR in the tears of 16 (47.1%) of 34 chronic hepatitis patients, 2 of 5 asymptomatic carriers, and on the Maclakof tonometer. HBV DNA on the contact tonometer was not completely removed by wiping twice with alcohol cotton balls, and the authors suggest flushing by running tap water for 10 seconds and immersion in 2% glutaraldehyde for 10 minutes.

Carrier State↗
PubMed1995

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis.

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.

Alcaligenes↗
PubMed2003

An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.

Animals↗
PubMed2004

The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors.

Contamination of food and water supplies by microorganisms such as Escherichia coli, the need for point-of-care bedside analysis of biological samples, and concerns about terrorist attacks using biological organisms, have made the development of fast, reliable, and sensitive analytical methodologies for use in monitoring of pathogens very important. With a variety of biosensors being developed for extremely sensitive and rapid nucleic acid diagnostics, it has become even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample preparation. The application of ultrasound has the potential to create DNA fragments from genomic material with lengths that are suitable for determination using biosensors and microarrays. For example, application of 85 W power at a frequency of 20 kHz can produce a preponderance of fragments of 100-400 base pairs (bp) within several seconds, and sample processing can lead to over 75% conversion from genomic material to fragments in times of 20-30 s. A proportion of these fragments are in a single-stranded state and are suitable for hydridization with immobilized single-stranded DNA probe oligonucleotides using a fiber optic biosensor. Control of factors such as salt concentration, exposure time, ultrasound power, and the initial temperature of the solution, can affect the length and form (single- or double-stranded) of DNA fragments that are generated by ultrasound, and average fragment length can be adjusted by selection of these operating parameters.

Biosensing Techniques↗
PubMed1990

Development of an ELISA for the detection and determination of contaminating proteins in recombinant DNA derived human erythropoietin.

An enzyme linked immunosorbent assay (ELISA) has been developed for the quantitative determination of the most probable contaminating proteins (MCP) of recombinant human Erythropoietin produced in the mouse fibroblast cell line C-127. The developed ELISA is a double polyclonal sandwich type immunoassay, which allows a quantitation of the MCPs in the range of parts per million. The polyclonal antibody, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference MCPs (a collection of the most probable protein contaminants) by immunoblot analysis and immunoabsorption of radiolabeled MCPs. Affinity purification of this antibody preparation using the immobilized MCPs resulted in an assay with higher signal-to-noise ratio. The assay was demonstrated to be very specific for the MCPs obtained from the rhu EPO purification process. Since the purification of each recombinant DNA derived protein expressed in mammalian cells requires its own unique process, no generic assay for contaminating proteins can be developed. There are only a few common criteria for the development of such multi-antigen ELISA, which will be discussed in this paper.

Animals↗
PubMed1981

Detection and elimination of cellular nucleic acids in biologicals produced on continuous cell lines.

Experiments have been conducted to determine the extent to which currently available purification techniques can remove contaminating substrate cellular DNA from inactivated poliovirus vaccine produced on continuous cell lines rising highly [32P]-labeled, nick-translated cellular DNA added to poliovirus suspensions, we found that purification procedures were capable, in small-scale experiments, of reducing contaminating DNA by factors of 10(3) (DNAse treatment followed by gel filtration) and 10(3)-3X10(5) (ion exchange chromatography). Sequential application of these purification steps should reduce contaminating cellular DNA to acceptable levels. We also examined the potential usefulness of immobilized nucleic acid hybridization techniques for the routine direct testing of residual cellular nucleic acids in final production lots of inactivated poliovirus vaccine and other biologicals. A filter hybridization test, using [32P]-labeled, nick-translated cellular DNA as a probe, was capable of detecting 40 pg of homologous cellular DNA. Using probes of higher specific activity the assay should be sensitive enough for use in routine quality control.

Animals↗
PubMed1993

A mouse model for the study of in vivo mutational spectra: sequence specificity of ethylene oxide at the hprt locus.

We have developed an approach for determining mutational spectra in exon 3 of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene in splenic T-lymphocytes of B6C3F1 mice. Hprt- mutants from treated animals were isolated by culturing splenic T-cells in microtiter dishes containing medium supplemented with IL-2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking the exon 3 region of hprt. Identification of samples containing mutant exon 3 sequences and purification of mutant DNA from contaminating wild-type hprt DNA was accomplished using denaturing gradient gel electrophoresis. Purified mutant sequences were then sequenced. This approach is being used to study the sequence specificity of ethylene oxide (ETO). 12-day-old mice were given single i.p. injections of 100 mg ETO/kg every other day or 30, 60, 90 or 120 mg ETO/kg daily for 5 days to achieve different cumulative doses of this compound. In mice exposed every other day, cumulative doses of 200, 600 and 900 mg ETO/kg produced average mutant frequencies of 15 +/- 12.8, 45 +/- 13.2, and 73 (70, 75) x 10(-6), respectively, 8 weeks after the first treatment. In mice exposed daily, cumulative doses of 150, 300, 450 and 600 mg ETO/kg produced average mutation frequencies of 4.2 +/- 10.4, 8.2 +/- 10.4, 11.1 +/- 1.0 and 15.5 +/- 10.7 x 10(-6), respectively, 20 weeks after the first treatment. The mutant fraction in control mice was less than 3 x 10(-6). 123 hprt- mutants from mice exposed to 600 or 900 mg ETO/kg were isolated and analyzed for mutations in exon 3. 18 were located in exon 3 (14.6%). DNA sequencing revealed that 11/18 mutations were base-pair substitutions at 8 different sites in exon 3. Four AT transversions, three AT transitions, two GC transversions, and two GC transitions were observed. Three of the substitutions (2 AT-->CG, 1 AT-->GC) occurred at one base (203) in a single animal. The remaining 7 mutations, isolated from 4 different animals, were the same +1 frameshift mutation in a run of 6 consecutive guanine bases (207-212) in exon 3. These results suggest the involvement of both modified guanine and adenine bases in ETO mutagenesis. The mouse T-cell cloning/sequencing assay for hprt described here represents a useful system for studying the molecular mechanism of chemically-induced mutation occurring in vivo at an endogenous gene.

Animals↗