Development of a high throughput SNP typing method using capillary electrophoresis

单核苷酸多态性（Single nucleotide polymorphisms， SNP）是人类基因组中数量最多的DNA标记，已引起许多研究领域的关注。在本研究项目中，我们试图通过多重单核苷酸引物延伸反应和毛细管电泳构建SNP分型方法。利用该方法，我们可以建立ABO血型基因分型方法、HLA-DRB1基因分型方法、Y染色体多重snp分型方法以及实现每条染色体上37个snp分型的高效方法。所得结果如下：1。用该方法测定了103例日本人的ABO基因型，估计的等位基因频率与文献报道一致。我们将这种方法应用于个案样本，并成功地确定了骨骼、牙齿、肌肉、器官、指甲和精液污染的阴道内容物的ABO基因型。我们检测了159名日本男性Y染色体上15个不同的SNP位点，检测到13个单倍型。各位点的基因多样性范围为0.025 ~ 0.486，单倍型多样性估计为0.838。该方法可用于个人鉴定和专利鉴定。本方法可建立HLA-DRB1基因分型方法，特别适用于混合标本的分析。我们建立了一种有效的方法来实现每条染色体上的37-plex分型。该方法具有较高的鉴别能力，有望成为法医标本分析的有效手段。

Single nucleotide polymorphisms (SNP), the most numerous DNA markers in the human genome, have attracted the attention in many research fields.In this research project, we have attempted to construct the method for SNP typing by a multiplexed single nucleotide primer extension reaction and capillary electrophoresys. Using this method, we could establish a method for genotyping of ABO blood groups, a method for genotyping of HLA-DRB1, a method for multiplex SNPs typing of Y chromosome, and an efficient method which realizes 37-plex typing of SNPs on each chromosome. The results obtained were as follows.1. ABO genotypes of 103 Japanese could be determined by this method, and the estimated allele frequencies were agreed with the previous reports. We applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal contents.2. We examined 15 different SNP loci on Y chromosome in 159 Japanese males, and detected 13 haplotypes. Gene diversity for each locus ranged from 0.025 to 0.486 and the haplotype diversity was estimated to be 0.838. This method could be applied for personal identification and patenity testing.3. We could establish a method for genotyping of HLA-DRB1 by the present method, and the method was useful particularly in the analysis of mixed specimens.4. We have establish an efficient method which realizes 37-plex typing of SNPs on each chromosome. This method holds promise as an effective means for analyzing forensic specimens and provides us with high discrimination power.