Establishment of MNSs haplotyping methods by gene analytical techniques.

（1）MNSs血型基因分型的研究本研究探索了用PCR方法对MN和Ss血型系统进行简单、准确的分型，建立了用限制性片段长度多态性（RFLP）或单链构象多态性（SSCP）分析进行MN基因分型的方法和用等位基因特异性PCR扩增进行Ss基因分型的方法。在此基础上，对SSCP分析的实验条件进行了研究，发现了几个可能存在MN血型基因重复等位基因的DNA样品，并对该基因的其他多态位点进行了分析。（2）单倍型分型方法的探索和研究在上述研究中，发现MN血型基因的常规M等位基因分为两个亚等位基因MG和MT，并评估了它们在日本人、中国北方和南方人群中的等位基因频率。为了建立分析由不同多态性基因如MN和Ss基因组成的单倍型的方法，首先应用简单的等位基因特异性反向PCR技术检测由MG、MT和N组成的MN系统的基因内单倍型。应用反向PCR技术检测ABO血型系统基因内单倍型，其中ABO基因多态性位点间的距离远大于MN基因多态性位点间的距离，目前正在进一步研究建立MNSs（intergene）单倍型分析方法。

(1)Investigations on genotypilg MNSs blood group Simple and accurate methods to genotype MN and Ss blood group systems by PCR were investigated, and the methods for MN genotyping using restriction fragment length polymorphism or single strand conformation polymorphism (SSCP) analysis, and the method for Ss genotyping using allele-specific PCR amplification were established and reported. Experimental condition for SSCP analysis was then studied and reported as a research manuscript.Several DNA samples which may involve duplicated alleles of MN blood group gene were found during the studies, and other polymorphic sites in the gene have been searched to analyze the duplicated allele.(2)Exploration and investigation of haplotyping methodsDuring the studies described above, conventional M allele of MN blood group gene was found to be divided into two sub-alleles, MG and MT, and their allele frequencies in Japanese, northern and southern Chinese populations were evaluated. To establish the method to analyze haplotypes consisting of different polymorphic genes such as MN and Ss genes, a simple allele-specific inverse PCR technique was applied first to detect an intragene haplotype of MN system consisting of MG, MT, and N.This technique (inverse PCR technique) was applied to detect an intragene haplotype of ABO blood group system, in which distance between polymorphic sites in ABO gene was much greater than that in MN gene.Further investigation is being continued to establish the method to analyze MNSs (intergene) haplotype.