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

基于反向PCR技术检测血型基因多态性核苷酸变异的新方法研究

基本信息

批准号：
11670428
负责人：
AKANE Atsushi
金额：
$ 2.05万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

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.

我们发现MN血型系统的M等位基因可分为M^G和M^T两个等位基因，并探索了一种等位基因特异性反向PCR扩增（ASIP）方法，以同时检测基因中的两个多态性区域。对血型糖蛋白A基因进行测序后发现，这3个等位基因可分为6个以上的等位基因，并对等位基因的分子进化进行了探讨。本文还研究了一种新的ASIP方法，主要等位基因A^1、B和O^1是根据ABO基因外显子6和7的碱基变化确定的，因此，ABO基因型的确定应单独检测这两个区域。本研究采用反向PCR技术，将ASIP、限制性片段长度多态性和测序方法同时检测分离的多态性区域。此外，这些方法只用于检测变异等位基因A^2，而对基因型A_2B的分型信息量很小，因为没有包括O等位基因<fus>。Se^<fus>等位基因是Se 1假基因和Se 1基因的融合基因。然而，我们通过对融合基因进行测序，揭示了融合机制的一些新发现。在此基础上，我们还探索了等位基因特异性PCR扩增和ASIP方法对Se血型进行基因分型。然后我们发现，在反向PCR之前，用绿豆核酸酶使片段末端变平对于片段的分子内连接是相当有效的。通过该方法，可以提高基于反向PCR的基因分型方法的精确度和适用性。