Gm genotyping from forensic biological materials by DNA hybridization

采用聚合酶链式反应(PCR)技术对免疫球蛋白G(Ig G)和免疫球蛋白kappa轻链(Ig Kappa)基因的遗传多态性进行了研究。该方法首先对提取的人基因组DNA进行第一次聚合酶链式反应(PCR)扩增免疫球蛋白基因内的多态区，然后对其中的一小部分扩增产物进行二次扩增，利用等位基因特异性引物对免疫球蛋白基因进行分型。用这种方法对免疫球蛋白基因恒定基因上的等位基因G3m*t和非G3m*t等位基因进行了分析。分别测定了G3m(T)等位基因及其对偶等位基因Non-G3m(T)，并对Km*1、Km*1.2和Km*3等位基因进行了分型。以第一次聚合酶链式反应的产物为样本的第二次聚合酶链式反应可以清楚地识别三个等位基因之间的序列变化。对冈山县72人的微小群体研究结果表明，Km*3和Km*1.2的基因频率分别为0.736和0.264。在检测G3m(T)恒定基因的情况下，第一次聚合酶链式反应成功地从样本DNA中扩增出含有G3m(T)异型多态区的726bp片段。以第一次聚合酶链式反应的产物为样本，第二次聚合酶链式反应基本上可以区分两个等位基因之间的核苷酸替换。

We have investigated the genetic polymorphisms of immunoglobulin G (IgG) and immunoglobulin kappa light chain (Ig kappa) genes by using the polymerase chain reaction (PCR).In the present methods, samples of extracted human genome DNA were subjected to the first PCR for the amplification of the polymorphic regions within the immunoglobulin genes.Small portions of the amplification products obtained from the first PCR were further subjected to the second PCR amplifications in which the allele specific primer sets were used for genotyping of the immunoglobulin genes.By this methods, alleles on the IgG3 constant gene, G3m*t andnon-G3m*t alleles, which determine the G3m(t) allotype and its antithetical isoallotype non-G3m(t), respectively, and alleles on the Ig kappa constant gene, Km*1, Km*1.2 and Km*3, were examined for genotyping.For the examination of the Ig kappa constant gene, 353bp fragments which contain the polymorphic region of the Km allotypes were successfully amplified from sample DNAs by the first PCR. The second PCRs using the products of first PCR as samples could clearly identify the sequence changes between the three alleles. Results of a tiny population study among 72 individuals living in Okayama Prefecture showed the gene frequencies of Km*3= 0.736 and Km*1.2= 0.264.In the case of the examination of the IgG3 constant gene, 726bp fragments which contain the polymorphic region of the G3m(t) allotype were successfully amplified from sample DNAs by the first PCR. Using the products of first PCR as samples, the second PCRs could generally distinguish the nucleotide substision between the two alleles.