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 o rigin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for indivi dualization of biological samples have reached a new boundary with the appl ication of the polymerase chain reaction (PCR) in DNA profiling, but a succ essful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investi gated the influence of storage conditions, sample volume, concentration mod es, extraction procedures, and chemical preservations on the quantify of DN A recovered, as well as the success rate of PCR-based genotyping for DQA1 a nd PM loci in urine. Urine specimens from male and female Volunteers were d ivided and stored at various temperatures for up to 30 days. The results su ggested that sample purification by dialfiltration, using 3000-100,000 mole cular weight cut-off fillers, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DN A by the organic method and by the resin method gave comparable typing resu lts. Larger sample volume yielded a higher amount of DNA, but the typing ra tes were not affected for sample volumes between 1 and 5 ml. The quantifiab le amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples rind decreased with the elapsed time und er both loom temperature (RT) and frozen storage. Typing of the male sample s also demonstrated that RT storage samples produced significantly higher s uccess rates than that of frozen samples, while then was only marginal diff erence in the DNA typing rates among the conditions tested using female sam ples. Successful assignment of DQA1+PM genotype was achieved fur all sample s of fresh urine, independent of gender, starting sample volume, or concent ration 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 dura tion, freezing at -70 degrees C may be more appropriate. Thus, the applicab ility of the DQA1+PM typing was clearly demonstrated for individualization of urine samples. (C) 1999 Elsevier Science Ireland Ltd. All rights reserve d.