| Summary: | 博士 === 國立清華大學 === 生命科學系 === 90 === Abstract
Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. However, BQ-chewing is also a risk factor for oral diseases and/or cancers. Although archived medical oral-specimens can be used to identify specific individuals under adverse conditions, STR markers are known to be unstable in various tumor tissues. On the other hand, 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.
The stability of forensic marker systems of both sequence and length polymorphism was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy betel quid (BQ) chewers and the archived clinical specimens from oral cancer patients.
The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, and corresponding peripheral blood samples from the same subject. A group of 100 non-BQ-chewers were used as the control. The genotypes of oral and paired peripheral blood samples were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTRTM markers (including 9 STR loci and the Amelogenin gene). As for the development of human DNA extracted 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.
In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalances, and allelic alterations including expansion, contraction and the un-classified types (can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (5 patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found in 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, 5 had the contraction, and 3 had the un-classified type. Further, among these 17 patients, 3 were found to acquire multiple allelic alterations at multiple loci. For the DNA marker of sequence polymorphism, one discordant result at D7S8 was found in the 600 DQA1/PM-marker loci. The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed with both DQA1/PM and STR marker systems, although two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, x2, and Scheffe tests indicated that the means of allelic imbalance at the 9 STR loci of the oral cancerous group revealed a significant difference from those in the control group.
The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers’ oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.
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.
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