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

在本研究的前期工作中，我们构建了两个新的常染色体STR基因座多重分型系统。这些贡献发表在期刊上。其中一个是专门设计的，具有高灵敏度的系统。为了筛选具有较高鉴别力的基因座，对两个日本地区的DNA样本进行了14个Y-STR基因座的单倍型分析（207例在本州，87例在冲绳）和117例泰国样本使用10重（DYS 436、DYS 439、DYS 435、DYS 19、DYS 460、Y-GATA-H4、DYS 391、DYS 392、DYS 438、DYS 437）和市售的Y-PLEX 6试剂盒（DYS 393、DYS 19、DYS 389、DYS 390、DYS 391、DYS 385），因为在两个系统中具有共同的两个基因座（DYS 19和DYS 391）。在此基础上，我们构建了两种高灵敏度的DNA多重检测体系。一个是用三色荧光染料标记的三个基因座（DYS 392、DYS 438和DYS 393），另一个是用三色荧光染料标记的四个基因座（DYS 19、DYS 3891、DYS 438和DYS 460）。后者是特别有用的，因为它使得有可能在通常的PCR循环数（30个循环）下从50 pg模板DNA和通过增加循环数从更少量的模板分型4个基因座。由于还计算了94个日本男性样本的单倍型多样性，并且该值非常高，为0.972，因此从区分能力的角度来看，该系统在法医学领域也是非常有用的。这些结果将在不久的将来提交其他地方。在此基础上，实现了本课题研究的目的.

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.