Investigation of novel methods detecting separated polymorphic nucleotide variations in blood group genes based on the inverse-PCR technique

点击翻译按钮获取中文摘要

We found that M allele of MN blood group system can be divided into two alleles M^G and M^T, and explored an allele-specific inverse-PCR amplification (ASIP) method to detect two polymorphic regions in the gene simultaneously. After the sequencing of glycophorin A gene, we found that the three alleles can be classified into more than 6 alleles, and discussed molecular evolution of the alleles. A new ASIP method was also investigated to detect these alleles.Major alleles, A^1, B and O^1 are based on the base changes at exsons 6 and 7 of ABO gene, so that the ABO genotype should had been determined by detecting both regions individually. In this study, the separated polymorphic regions could be detected simultaneously by ASIP, restriction fragment length polymorphism and sequencing methods with inverse-PCR technique. Moreover, these methods were applied to detect a variant allele A^2, but were little informative to type genotype A_2B because no O allele was included.Major alleles of Se blood group in Japanese are Se1, sej and se^<fus>. The se^<fus> allele was reported to be a fusion gene of Sec1 pseudogene and Se1 gene. However, we revealed several new findings on the fusion mechanism by sequencing the fusion gene. Based on the findings, we also explored the allele-specific PCR amplification and ASIP methods to genotype Se blood group. Then we found that flattening of the fragment ends by mung bean nuclease was quite effective for intramolecular ligation of the fragments prior to the inverse-PCR. By this procedure, the precision and applicability of the inverse-PCR-based genotyping methods could be increased.