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

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聚合酶链式反应(ASIP)同时检测该基因中两个多态区域的方法。对血糖素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，并对这些等位基因的分子进化进行了讨论。主要等位基因A^1、B和O^1是基于ABO基因外显子6和7的碱基变化而来的，因此ABO基因型的确定应通过分别检测这两个区域来进行。在本研究中，利用ASIP、限制性片段长度多态性和反向PCR测序技术可以同时检测到分离的多态区域。此外，这些方法还被用于检测变异等位基因A^2，但由于不包含O等位基因，因此对A2B型的信息量很小。日本人Se血型的主要等位基因是SE1、Sej和Se^&lt；fus&gt；Se^&lt；fus&gt；等位基因被认为是Sec1假基因和SE1基因的融合基因。然而，我们通过对融合基因的测序揭示了关于融合机制的几个新发现。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血型进行分型。然后我们发现，绿豆核酸酶平整片段末端对于反向PCR之前的片段的分子内连接是非常有效的。通过该方法，可以提高反向聚合酶链式反应基因分型方法的准确性和适用性。

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.