Establishment of a high throughput single-nucleotide-polymorphisms (SNPs) screening system based on the re-annealing and digestion reaction

Se血型在法医学领域具有重要意义，因为它与体液中ABO血型抗原的分泌有关。在这项研究中，首先，Se 1，Se 2，sej和se等位基因的Se血型，这是常见的日本人口的基因型，使用温度梯度凝胶电泳方法。杂合样本中出现的异源双链带显示出基因型特异的电泳图谱。当存在一个错配核苷酸时，在凝胶中发现一条或两条异源双链带。在有两个碱基错配的样品中，观察到四条异源双链带。这些结果表明，即使在分析未知等位基因时，该方法也可以估计错配核苷酸的数量。分析A、B、O^和O G四个典型等位基因的第6和第7外显子时<A->，由于GC含量的差异，同源双链带显示等位基因特异性电泳图谱。不同等位基因混合后可检测到不同的异源双链带，从而可用于ABO血型的基因分型，并将该方法应用于线粒体DNA高变区1（mtDNAHV1）的鉴定。mtDNA HV1的同源双链带显示不同的电泳图谱，不同样品的混合物显示特异的异源双链带，因此该方法可用于mtDNA HV1变异体的鉴定，而异源双链带中的错配核苷酸不能用文献报道的方法切割。因此，温度梯度凝胶电泳是检测等位基因和变异体单核苷酸多态性（SNPs）的最合适的高通量筛选方法。

Se blood group is important in the forensic field because it is associated with the secretion of ABO blood group antigens in body fluids. In this study, first, Se1, Se2, sej and se alleles of the Se blood group which are common in Japanese population were genotyped using the temperature-gradient gel electrophoresis method. Heteroduplex bands occurred in the heterozygotic samples showed genotype-specific electrophoresis patterns. When one mismatched nucleotide existed, one or two heteroduplex bands were found in the gel. In the samples with two mismatched bases, four heteroduplex bands were observed. These findings suggested that numbers of the mismatched nucleotides could be estimated even when an unknown allele was analyzed by the method.This method was applied to the genotyping of ABO blood group. When exons 6 and 7 of the four typical alleles, A, B, O^<A-> and 0G were analyzed, the homoduplex bands showed allele-specific electrophoresis patterns due to difference in the GC contents. Different heteroduplex bands could be detected after mixture of different alleles, so that ABO blood group could be genotyped by this method.This method was also applied to identify the htpervariable region 1 of mitochondrial DNA (mtDNA HV1). Homoduplex bands of mtDNA HV1 showed different electrophoresis patterns, and mixture of different samples revealed specific heteroduplex bands, so that mtDNA HV1 variants could be identified by this method.Mismatched nucleotides in heteroduplex bands could not be cleaved by means of the reported techniques. So, the temperature-gradient gel electrophoresis was the most suitable, high throughput screening method to detect single nucleotide polymorphisms (SNPs) in alleles and variants.