The selection of new microsatellite loci suitable for fortune forensic exam ination and the establishment of its wide use

在本研究项目（2年）期间，我们使用MapPair 8A （research Genetics Inc.）的STR引物进行连锁分析，分析了人类基因组中168个平均间距为25 cM的STR位点。这套引物包括168对引物，每对引物中有一对用三色荧光染料（FAM， HEX， TET）标记。DNA样品扩增，PCR产物用遗传分析仪310按8A多效表电泳，每个样品根据分级结果进行分型。我们已经用32个日本人的DNA样本分析了全部168个基因座。在杂合度大于0.8的20个STR基因座中，选择了序列结构简单的12个STR基因座。这些基因座的序列在本研究中得到了准确的确认，并进行了轻微的修正。我们尝试用三色荧光染料构建2个多重体系，每6个STR位点。我们为新系统重新设计了每个位点的引物。一个系统完成了，另一个也差不多完成了。此外，我们还从诺丁汉大学Armour教授分离的约20个微卫星基因座中选择了具有较高杂合性且无不规则等位基因的3个STR基因座（wglc5、wgld1、pq2h2），设计了一个新的多重系统。该研究结果在日本DNA多态性研究学会第八届年会上发表，包括在法医领域的有用性。这样，新的微卫星分型系统已基本完成，该系统使用15个位点，适用于日文法医检查。

Within the term of this research project (2 years), we analyzed 168 STR loci spacing at 25 cM on average in human genome using a STR primer set for linkage analysis, a MapPair 8A (Research Genetics Inc.). This primer set includes 168 primer pairs in which one of each pair is labeled with either of the tricolor fluorescent dyes (FAM, HEX, TET). DNA samples were amplified and the PCR products were electrophoresed according to the Version 8A multiplex table with the Genetic Analyzer 310, and each sample was typed from sizing results. We have finished to analyze all 168 loci with 32 Japanese DNA samples. Out of 20 STR loci showing heterozygosities of higher than 0.8 twelve STR loci were selected because their sequence structures were simple based on the databases, GDB etc. The sequences of those loci were confirmed accurately in this study with minor corrections.We tried to construct 2 multiplex systems with each 6 STR loci using the tricolor fluorescent dyes. We newly designed each primer set at each locus for the new systems. One system was completed and another has almost been completed. Moreover, we have also devised a mew multiplex system using 3 STR loci (wglc5, wgld1, pq2h2) with comparatively high heterozygosity and without irregular allele selected from approximately 20 microsatellite loci which Prof. Armour in Nottingham University had isolated. The results including the usefulness in the forensic field were presented at the 8th annual meeting of the Japanese Society for DNA polymorphism Research.In this way, the new microsatellite typing systems using 15 loci appropriate to forensic examination in Japanese has almost been completed.