United States Department of Transportation
i
Urinary Genotyping for DQA1 and PM Loci Using PCR-Based Amplification: Effects of Volume, Storage Temperature, Preservatives, and Aging on DNA Extraction and Typing
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1999-04-01
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Edition:Final Report
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Abstract: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 biosamples 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 volumes, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based urinary genotyping for DQA1 and PM loci. 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 3,000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate 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 higher amount of DNA, but the typing rates were not affected for sample volumes between 1 to 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 sampling 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°C during a period of 30 days. For longer storage duration, freezing at -70°C may be more appropriate. Thus, the applicability of the DQA1+PM typing was clearly demonstrated for individualization of the urine samples.
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