Establishment of forensic molecular genetic analyzing method using the rapid detection allele-specific MVR-PCR

采用聚合酶链反应（MVR-PCR）在D1S8 （MS32）位点设计了一种新的等位基因特异性小卫星变异重复定位系统，无需southern blot杂交。在侧翼区域的3个多态性位点（H1、Hf和H2），采用a型的dCTP [R110]、T型的dUTP [R110]和3组等位基因特异性引物（H1G/C、Hf2+/-和H2C/T）进行等位基因特异性MVR-PCR。利用引物H2C、H2T和Hf2-，可以正确绘制出MVR阶梯的50个级距。即使在最远的多态性位点（H1位点，距离第一次重复约400 bp），引物H1C也可以显示大约40个MVR阶梯。而在使用引物Hf2+的情况下，只观察到微弱的梯级，因为引物可能被消耗用于二聚体的形成，引物二聚体成为主要产物。利用引物H1G，在第20阶附近出现了一个非特异性的宽带，干扰了进一步的定位。为了提高MVR的制图效果，我们设计了新的引物。通过对各引物序列的计算机分析，发现引物H1G与引物32-TAG-A/T形成引物二聚体，而引物Hf2+与引物TAG相互作用的可能性较大。因此，我们设计了两个新的引物，分别命名为H1G20A和Hf2+15C，通过替换引物中间的一个碱基来防止引物二聚体的形成，并保持等位基因的特异性。通过这些引物，我们可以正确地映射到35到40个梯级。此外，利用标准侧翼引物（32-D），我们不仅获得了足够的等位基因编码，还获得了45个以上的二倍体编码。这种新的MVR分析方法可应用于法医和群体遗传学领域。

A novel assay system was devised for allele-specific minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) at D1S8 (MS32) without southern blot hybridization. At three polymorphic sites (H1, Hf and H2) in the flanking region, allele-specific MVR-PCR was performed using dCTP [R110] for a-type, dUTP [R110] for t-type, and three sets of allele specific primers (H1G/C, Hf2+/- and H2C/T). Using primers H2C, H2T and Hf2-, about 50 rungs of the MVR ladder could be correctly mapped. Even though in the most distant polymorphic site (H1 site, approximately 400 bp from the first repeat), primer H1C made it possible to show about 40 rungs of the MVR ladder. However, in case of using primer Hf2+, only faint rungs were observed because the primer was probably consumed for dimer formation and the primer dimer became the predominant product. Using primer H1G, a non-specific broad band appeared around the position of 20th rung and disturbed further mapping. In order to improve MVR mapping results, we designed new primers. As the result of investigating the sequences of each primer by computer analysis, primer H1G was indicated to form a primer dimer with primer 32-TAG-A/T, while we found the strong possibility of primer Hf2+ to interact primer TAG.Therefore, We designed two new primers named H1G20A and Hf2+15C by replacing a base in the middle of each primer to prevent primer dimer formation and to remain the allele specificity. We could correctly mapped up to 35 and 40 rungs by these primers. Additionally, we obtained not only enough allele codes but also more than 45 diploid codes using the standard flanking primer (32-D). This novel method for MVR analysis can be applied to the field of forensic and population genetics.