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

设计了一种新的检测系统，利用聚合酶链反应（MVR-PCR）在D1 S8（MS 32）处进行等位基因特异性小卫星变异重复定位，而无需Southern印迹杂交。在侧翼区的3个多态性位点（H1、Hf和H2），使用dCTP [R110]（a型）、dUTP [R110]（t型）和3套等位基因特异性引物（H1 G/C、Hf 2 +/-和H2 C/T）进行等位基因特异性MVR-PCR。用引物H_2C、H_2T和Hf ~（2-）可正确定位MVR梯状条带的50个梯级。即使在最远的多态性位点（H1位点，距第一个重复约400 bp），引物H1 C也可以显示约40个MVR梯状条带。然而，在使用引物Hf 2+的情况下，仅观察到微弱的梯级，因为引物可能被消耗用于二聚体形成，并且引物二聚体成为主要产物。用引物H1 G在第20横档附近出现一条非特异性的宽带，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。通过计算机序列分析，发现引物H1 G与引物32-TAG-A/T形成引物二聚体，而引物Hf ~（2+）与引物TAG相互作用的可能性很大。我们设计了两条新的引物，命名为H1 G20 A和Hf 2 +通过替换每个引物中间的碱基以防止引物二聚体形成并保持等位基因特异性，从而使等位基因与15 C的等位基因特异性结合。这些引物可以正确定位35和40个梯级。此外，我们使用标准侧翼引物（32-D）不仅获得了足够的等位基因编码，而且还获得了45个以上的二倍体编码。这种新的MVR分析方法可以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.