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。本研究首先采用温度梯度凝胶电泳法对日本人群中常见的Se血型的Se1、Se2、Sej和Se等位基因进行了基因分型。杂合子样本中出现的异源双链条带表现为基因型特异性的电泳谱。当存在一个不匹配的核苷酸时，凝胶中出现一条或两条异源双链条带。在两个碱基错配的样品中，观察到4条异源双链条带。这些发现表明，即使在分析未知等位基因的情况下，也可以估计错配核苷酸的数量。该方法被应用于ABO血型的基因分型。对A、B、O^&lt、A-&gt和0g四个典型等位基因的第6、7外显子进行分析时，由于GC含量的不同，同源双链条带呈现出等位基因特异性的电泳带。将不同等位基因混合后可检测出不同的异源双链条带，可用于ABO血型的基因分型，并用于线粒体DNA hv1基因的hTV1区的鉴定。线粒体DNA HV1的同源双链条带表现出不同的电泳谱，不同样品的混合物显示出特定的异源双链条带，因此该方法可以识别mtDNA HV1变异体，但不能用已报道的技术切割异源双链条带中的错配核苷酸。因此，温度梯度凝胶电泳法是检测等位基因和变异体中单核苷酸多态(SNPs)的最合适的高通量筛查方法。

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.