Establishment of high-sensitive multiplex typing systems for Y-STRs and its application to a very small amount of specimens

在本研究的初步步骤中，构建了两个新的常染色体STR基因座多重分型系统。这些投稿都发表在这些期刊上。其中一个是专门为系统的高灵敏度而设计的。为了选择具有较高识别力的基因座，利用美国NIST设计的10-plex(DYS436、DYS439、DYS435、DYS19、DYS460、Y-GATA-H4、DYS391、DYS392、DYS438、DYS437)和市售的DYS393、DYS19、DYS389、DYS390、DYS391、DYS385试剂盒，对日本的两个地方DNA样本(本州207份和冲绳87份)和117份泰国样本的14个Y-STR基因座的单倍型进行了分析。在这些研究的基础上，我们完成了两种针对高度降解DNA样本的高灵敏多重体系的构建。一个是用三色荧光染料标记的三个基因座(DYS392、DYS438和DYS393)，另一个是用三色荧光染料标记的四个基因座(DYS19、DYS3891、DYS438和DYS460)。后者特别有用，因为它使得在通常的PCR循环数(30个循环)下从50pg的模板DNA中分型4个座位成为可能，并且通过增加循环数从较少的模板中分型成为可能。同时还计算了94份日本男性样本的单倍型多样性，其值很高，为0.972，从鉴别力的角度来看，该体系在法医领域也有很大的应用价值。这些结果将在不久的将来提交到其他地方。通过这笔资金，我们构建复接系统的研究已经达到了目标。

In the preliminary step toward the present study, two novel multiplex typing systems for autosomal STR loci were constructed. These contributions were published in the journals. One of them was specially designed to have high sensitivity of the system. In order to select the loci with higher discriminating power, the haplotypes at 14 Y-STR loci were analyzed from two regional Japanese DNA samples (207 in Honshu and 87 in Okinawa) and 117 Thai samples using the 10-plex (DYS436, DYS439, DYS435, DYS19, DYS460, Y-GATA-H4, DYS391, DYS392, DYS438, DYS437) devised by NIST in the USA and the commercially released Y-PLEX6 kit (DYS393, DYS19, DYS389, DYS390, DYS391, DYS385) because of having two loci (DYS19 and DYS391) in common in both systems. Based on these studies, we completed to construct two kinds of high-sensitive multiplex systems for highly degraded DNA samples. One was for three loci (DYS392, DYS438 and DYS393) labeled with three-colored fluorescent dyes and another was for four loci (DYS19, DYS3891, DYS438 and DYS460) labeled with three-colored ones. The latter was especially useful because it made it possible to type the 4 loci from 50 pg of template DNA at usual numbers of PCR cycles (30 cycles) and from less amount of the template by increasing the number of cycles. Since the haplotype diversity was also calculated from 94 Japanese male samples, and the value was very high with 0.972, it was indicated this system could be very useful in forensic fields from the point of discriminating power as well. These results will be submitted elsewhere in near future. By this grant, our study to construct the multiplex system has been achieved to the goal.