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

在本研究项目的期限内（2年），我们使用用于连锁分析的STR引物组MapPair 8A（Research Genetics Inc.）分析了人类基因组中平均25cM间隔的168个STR基因座。该引物组包括168个引物对，其中每对中的一个用三色荧光染料（FAM、HEX、泰特）中的任一种标记。扩增DNA样品，并根据8A版多重表用遗传分析仪310对PCR产物进行测序，并根据大小测定结果对每个样品进行分型。我们已经完成了对32个日本DNA样本的全部168个位点的分析。从杂合度大于0.8的20个STR基因座中，根据数据库、GDB等数据库，筛选出12个序列结构简单的STR基因座，并对这些基因座的序列进行了准确的验证，尝试用三色荧光染料构建2个各6个STR基因座的复合体系。我们在新系统的每个位点重新设计了每个引物组。一个系统已经完成，另一个系统也几乎完成。此外，我们还从诺丁汉大学Armour教授分离的约20个微卫星位点中选出3个杂合度较高的STR位点（wglc 5、wgld 1、pq 2h 2），设计了一个新的复合扩增体系。在日本DNA多态性研究学会第8届年会上发表了包括在法医学领域的实用性在内的研究结果。这样，使用15个基因座的适用于日本法医学检查的新型微卫星分型系统几乎已经完成。

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.