Development on practical examination methods using DNA polymorphisms in the field of Forensic Dentistry

本研究的目的是建立实用可靠的DNA多态检测方法，利用高度降解的DNA来源检测口腔法医中的DNA多态。在第一年，我们检查了STS上PC大小的减小是否产生了可重复性的结果，以及它是否提高了从降解的DNA中扩增的效率。我们比较了D12SG6、VWA和CD4等小片段ST基因座扩增的可靠性，并考察了从同一多态区域产生不同大小的PCB产物的引物应用于PC时对分型的影响。结果表明，PC大小的减小提高了降解DNA的扩增效率，但并不协调分型的可靠性。我们调查了201名日本人线粒体DNA控制区的序列多态性，并根据邻近Joining分析的结果将165种单倍型划分为32个类群。每个簇共享特定的多态位点，这些特征表明mtDNA多态可能应用于从法医生物样本中进行种族群体评估。我们还调查了日本人群中的10个Y染色体ST多态，并支持了DYS390基因座的结构变异，从而确定了Y-ST多态具有高度信息量的区分力。BeausST多态有时会在降解DNA的PC中产生错误的结果，我们通过设计扩增小片段PC产物的引物来扩展我们对Y-SNPs的研究，这使得从高度降解的DNA中获得扩增产物成为可能。我们还检查了常染色体ST多态和SNPs，以比较从降解的DNA样本中分型的可靠性。Y-SNPs的单倍型分析表明，在日本人群中男性个体中存在多个谱系。我们还展示了患者使用的操作程序和树脂。

The purpose of this study is to establish practicable and reliable methods to detect DNA polyomorphisms using highly degraded DNA sources in Forensic Dentistry. In the first year, we examined if reduction of the PC size on STs gave reproducible results and also if it improved amplification efficiency from degraded DNAs. We compared reliability of amplification of the small-sized ST loci, such as D12SG6, VWA and CD4 and examined influences on typing when the primers producing different-sized PCB products from the same polymorphic region were constructed and applied to the PC. The results indicated that reduction of the PC size increased amplification efficiency from degraded DNAs, but did not coordinate reliability of typing. We investigated sequence polymorphism of mitochondrial DNA control regions from 201 Japanese individuals and classified 165 haplotypes into 32 clusters based on the results of Neighbour Joioning analysis. Each clusters shared specific polymorphic sites, and these characteristics suggested possible application of mtDNA polymorphisms to ethnic group estimation from forensic biological samples. We also investigated 10 Y chromosome ST polymorphisms in the Japanese population and also shoed structural variation in the DYS390 locus, resulting in the identification of highly informative discriminatory power of Y-ST polymorphisms. Beauses ST polymorphisms sometimes gave erroneous results in PC of degraded DNA, we extended our studies on Y-SNPs by designing primers to amplify small PC products, which made it possible to obtain amplification products from highly degraded DNAs. Autosomal ST polymerphisms and SNPs were also examined to compare reliability of typing from degraded DNA samples. Haplotype analysis on Y-SNPs indicated the presence of several lineages in male individuals in the Japanese population. We also showed procedure and resin used by the patients.