Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.
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.
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Toxicology and Accident Research Laboratory, Federal Aviation Administration, U.S. Department of Transportation, Oklahoma City, OK 73125-5066, USA.
Chaturvedi
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Canfield
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D004247:DNA / Q000145:classification / Q000235:genetics / Q000652:urine*; D005260:Female; D005819:Genetic Markers; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics* / Q000652:urine; D059848:HLA-DQ alpha-Chains; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D013048:Specimen Handling / Q000379:methods*
D005819:Genetic Markers; D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D011933:Reagent Kits, Diagnostic; D004247:DNA