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A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFlSTR Profiler Plus PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris-HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFlSTR Profiler Plus STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The number of matching alleles varied between not less than 10 for one donor to no more than two for another donor. These results may well be linked to the environmental experience of the hairs from each donor prior to removal.
Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.
Two species of parasitic wasp, Venturia canescens and Leptomastix dactylopii, were killed and preserved by various methods used for Hymenoptera and in mass-collecting devices. Total genomic DNA was subsequently extracted and a 524 bp fragment of the mitochondrial 16S ribosomal RNA gene amplified by PCR. Results for these techniques were compared with that for fresh material and museum specimens. Material from -80 degrees C, 100% ethanol, air-drying in a desiccator, and critical-point dried from alcohol all yielded good results after short and long-term storage, as did specimens from ethylene glycol but not formalin (the latter two being commonly used in pitfall and flight intercept traps). Specimens killed in ethyl acetate vapour and air-dried yielded very degraded DNA which did not successfully PCR. The use of this killing agent is a likely reason for previous reports of inconsistent results obtained from museum specimens, and the now widespread use of critical-point drying of wasps and other insects from alcohol is advocated as a potential source of DNA from rare taxa.
N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99 +/-20.3)x10(-7) versus (0.58+/-0.50)x10(-7) N7-MedGp/deoxyguanosine-3'-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59+/-15.6)x10(-7) N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1+/-38.0)x10(-7) versus (2.73+/-4.07)x10(-7) N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5+/-13.1)x10(-7) versus (2.64+/-3.89)x10(-7) N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.
BACKGROUND: The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set. RESULTS: Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers. CONCLUSION: These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.
The enzymatic amplification of specific DNA sequences by the polymerase chain reaction (PCR) has provided a new approach to genetic typing of HLA-class II region specificities. However, we found the existing PCR protocols to be adequate and/or time consuming, especially when large numbers of samples need to be processed. We have devised a simplified method which alleviates these problems. Genomic DNA prepared from peripheral blood nucleated cells was facilitated using the punched-out filter paper absorbed with the peripheral blood. Second exon HLA-DRB segments of the genomic DNA were amplified by the polymerase chain reaction (PCR) (30 cycles: denaturing at 94 C for 1 min; annealing at 55 for 1 min, extension at 72 C for 2 min) using HLA-DRB primer DRB 5'-1 (ACCGGTCGTTCITGTCCCCICAGCA) and DRB 3'-1 (CTCGCCICTGCACIGTIAAGC) designed in our laboratory. The presence of specific alleles in a PCR-amplified sample was determined by dot-blot hybridization with 32 P-labeled sequence-specific-oligonucleotides (SSOs). Quantitation of the producer is being evaluated using an automated scanner. These newly designed primers allowed amplification of all the known expressed alleles of DRB 1, B 3, B 4 and B 5 loci. This type of reproducible and precise assay is very useful in the HLA-class II typing of large number of patients.
Molecular methods are being frequently used for the study of soil microbial communities as majority of naturally occurring microbial populations are non-culturable. In the present study, we describe a protocol of DNA extraction from diverse soils using a combination of heat, enzyme (lysozyme) and guanidine thiocyanate. The efficacy of the procedure was evaluated in terms of yield, purity and duration of extraction. The protocol was effective for neutral, acidic as well as alkaline soils (pH range 4.5-8.5). The extracted soil DNA was observed with negligible shearing on agarose gel and the time taken for restriction digestion was very less. Further, the DNA extracted was almost completely devoid of contaminants and pure enough which could be used for PCR amplification and Southern hybridization.
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Organic-aqueous liquid (phenol) extraction is one of many standard techniques to efficiently purify DNA directly from cells. The cell components naturally distribute themselves into the two fluid phases in order to minimize interaction energies of the biological components with the surrounding solvents. The membrane components and protein partition to the interface between the organic and aqueous phases while the DNA stays in the aqueous phase. The aqueous phase is then removed with a purified DNA sample. This work studies the first steps towards miniaturizing this liquid extraction technique in a microfluidic device. The first step is to understand how the two liquid phases behave in microchannels. Due to the interfacial tension between the two liquid phases, novel approaches must be examined in order to obtain interfacial stability under flow conditions. The stability of the organic-aqueous interface is improved by reducing the interfacial tension between the two phases by incorporating a surfactant into the aqueous phase. The variation of the interfacial tension as a function of surfactant concentration is also quantified in this work. This has led to the ability to create stable stratified microflows in both a dual inlet and three inlet microfluidic systems. Also, the first step in understanding biological interactions at the organic-aqueous interface is investigated using a fluorescently labeled bovine serum albumin protein.
BACKGROUND: Nucleic acid testing (NAT) of pooled plasma samples from individual blood donations for viral nucleic acids has become widely established. Full automation of such sample processing can overcome many of the problems associated with methods used so far. STUDY DESIGN AND METHODS: In this study an automated extraction method for viral nucleic acids (parvovirus [PAV] B19 DNA, hepatitis B virus [HBV] DNA, and hepatitis A virus [HAV] RNA), starting directly from the minipool sample (n = 96, 9.6 mL), was evaluated. A magnetic separation module I (chemagic, Polymer Laboratories) in combination with the chemagic viral DNA and RNA kit special based on the use of magnetic beads was used for this purpose. More than 144 pools spiked with defined concentrations of reference material and an additional 102 pools negative for the analyte were extracted and amplified. The isolated viral nucleic acids were detected by polymerase chain reaction (PCR). RESULTS: The assays were highly specific and obtained a 95 percent detection limit of 875 IU per mL of pooled single donation for PAV B19, 260 IU per mL for HAV, and 1274 IU per mL for HBV, respectively. The crossing points showed variation coefficients from 1.49 to 2.76 percent. The turnaround time for the whole process was 3 hours. Testing of subpools to determine an infected single donation would be possible with the same general extraction method. A total of 102 unspiked minipools (96 x 100 microL per donation) were analyzed and none tested positive. CONCLUSION: The automated magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donations for viremic donors to further increase the safety of blood products. Minipools as well as subpools can be directly processed.
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A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.
Using a modified DNA mobility shift assay, we have identified and purified a novel 9-kDa polypeptide (designated p9) from plasmacytoma nuclear extracts which forms salt-stable DNA-protein complexes without apparent sequence specificity. In competition studies, p9 bound exclusively with naturally occurring DNA relative to RNA and preferentially with polydeoxypyrimidines among 10 homopolynucleotides tested. Nuclear extracts prepared from various murine and human cell lines contained a common factor which, when cross-linked with photoreactive [32P]DNA, co-migrated with covalent [32P]DNA-p9 complexes on polyacrylamide gels. An oligonucleotide, constructed on the basis of the N-terminal 29 amino acid residues of p9, was employed to isolate a 700-base pair cDNA clone encoding the complete polypeptide. On Northern blots, p9 cDNA hybridized with a mRNA species of comparable size from both mouse and human cell lines, suggesting a significant degree of interspecies sequence conservation. Amino acid and cDNA sequence analyses demonstrated that p9 derives from the 77-residue C-terminal domain of a 14-kDa polypeptide comprised of 127 amino acids. DNA binding activity was exhibited by peptides synthesized in vitro from run-off RNA transcripts corresponding to the truncated 9-kDa C-terminal domain, but not to the 14-kDa precursor, implicating proteolysis as a post-translational mechanism required for functional activation of p9.
Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender, starting sample volume, or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4 degrees C during a period of 30 days. For longer storage duration, freezing at -70 degrees C may be more appropriate. Thus, the applicability of the DQA1 + PM typing was clearly demonstrated for individualization of urine samples.