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

利用聚合酶链式反应(MVR-PCR)在D1S8(MS32)建立了一种新的无Southern杂交的等位基因特异性小卫星变异重复序列定位系统。在侧翼区的三个多态位点(H1、Hf和H2)上，分别使用a型的dCTP[R110]、t型的dUTP[R110]和三套等位基因特异性引物(H1G/C、Hf2+/-和H2C/T)进行MVR-PCR。使用引物H_2C、H_2T和H_f2-，可以正确地绘制MVR阶梯的大约50个梯级。即使在最远的多态位点(H1位点，距第一个重复序列约400bp)，引物H1c仍有可能显示约40个MVR梯级。然而，在使用Hf2+的情况下，只观察到微弱的条带，这是因为该引物可能用于形成二聚体，并且该二聚体成为主要产物。利用引物H1G，在第20级附近出现一条非特异的带状条带，干扰了进一步的定位。为了提高MVR定位结果，我们设计了新的引物。计算机分析结果表明，H1G与32-Tag-A/T形成一个二聚体，而Hf2+与TAG相互作用的可能性很大，因此，我们设计了两个新的引物H1G20A和HF2+15C，通过在每个引物中间替换一个碱基来防止二聚体的形成，同时保持了等位基因的特异性。通过这些引物，我们可以正确地绘制出多达35和40个梯级。此外，我们不仅获得了足够的等位基因编码，而且使用标准的侧翼引物(32-D)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学和群体遗传学领域。

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.