Extraction of human DNA for PCR from chewed residues of betel quid using a novel "PVP/CTAB" method

Residues of chewed betel quid (BQ) are often found on crime scenes in Taiwan and possibly some of the Southeast Asian countries. Although these residues are important biological evidences relating to the suspects, the forensic analysis of BQ evidence has been hindered by failures in extraction of human DNA for PCR analysis. Therefore, it is a prerequisite for relevant forensic casework to establish a reliable method for extracting DNA from chewed BQ residues. Three conventional methods (salt/chloroform, 5% Chelex-100 resin, and QIAamp) were first tested for extraction of human DNA from 33 mock BQ samples, which had been stored for less than two months, and 50 four-year-old forensic BQ samples. PCR amplifications from the HLA-DQA1&PM and the STR loci were then used to test the quality of the extracted DNA. For the mock samples, three observations were made. First, PCR amplification of DNA extracted by using these conventional methods had low success rate. Second, the addition of extra Taq DNA polymerase could compensate the lost enzyme activities due to putative inhibitors and, thus, increase the yield. Third, using the Centricon-100 column to remove putative inhibitors substantially improved the efficiency of PCR. However, for the four-year-old forensic BQ samples, none of the attempts for PCR were successful. In order to solve the problem in PCR analysis of DNA from old BQ samples, we developed a DNA extraction method based on the use of polyvinyl pyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), which bind to two common classes of PCR inhibitors in plants, polyphenols, and polysaccharides, respectively. The result showed that this "PVP/CTAB" method is completely successful for the mock BQ samples, and 92% (46 out of 50) successful for the four-year-old forensic BQ samples. To our best knowledge, this is the first report of a reliable method for the extraction of human DNA for PCR from chewed BQ residues. This method should provide a useful means for forensic identification in countries where betel chewing is common.