Yoshida M, Kimura A, Numano F, Sasazuki T
Department of Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Hum Immunol. 1992 Aug;34(4):257-66. doi: 10.1016/0198-8859(92)90025-i.
The polymerase chain reaction (PCR) in combination with the sequence-specific oligonucleotide probe (SSOP) was applied to analyze the polymorphism in the exon 2 of the HLA-B gene. In this study, genomic DNAs from 85 B-lymphoblastoid cell lines homozygous for HLA and peripheral blood granulocytes of 156 Japanese individuals were investigated. Two HLA-B-specific 5'-sided primers (CG4 and CG5) and two 3'-sided primers (CG2 and CG3) were designed for specific amplification of the exon. HLA-B alleles were classified into two groups (groups I and II) according to specific amplification with two types of the 3'-sided primers. The amplified DNAs were hybridized with 23 nonradioactively labeled SSOPs. Based on the hybridization patterns with the SSOPs, 34 HLA-B specificities were divided into 26 epitope combination (EC) groups. Fifteen HLA-B specificities were classified into four EC groups and these HLA-B specificities could not be distinguished from one another in the same EC group. Another 16 HLA-B specificities corresponded one by one to 16 distinct EC groups, and two subtypes of HLA-Bw75, B27, and Bw48 were also identified enabling the accurate typing of 22 HLA-B alleles at the DNA level. Single-strand conformation polymorphism (SSCP) of the PCR products from group I HLA-B alleles was also investigated. The HLA-B alleles showed distinct electrophoretic patterns in nondenaturing polyacrylamide gels, depending on the nucleotide sequences of the exon 2, indicating that the SSCP analysis may be an alternative, useful and practical HLA-matching system of HLA-B specificity in tissue transplantation.
将聚合酶链反应（PCR）与序列特异性寡核苷酸探针（SSOP）相结合，用于分析HLA - B基因第2外显子的多态性。在本研究中，对85个HLA纯合的B淋巴母细胞系的基因组DNA以及156名日本个体的外周血粒细胞进行了研究。设计了两条HLA - B特异性的5'端引物（CG4和CG5）和两条3'端引物（CG2和CG3）用于该外显子的特异性扩增。根据用两种3'端引物的特异性扩增情况，将HLA - B等位基因分为两组（I组和II组）。扩增的DNA与23种非放射性标记的SSOP进行杂交。根据与SSOP的杂交模式，将34种HLA - B特异性分为26个表位组合（EC）组。15种HLA - B特异性被分为4个EC组，并且在同一EC组内这些HLA - B特异性无法相互区分。另外16种HLA - B特异性一一对应于16个不同的EC组，还鉴定出了HLA - Bw75、B27和Bw48的两个亚型，从而能够在DNA水平上准确分型22种HLA - B等位基因。还研究了I组HLA - B等位基因PCR产物的单链构象多态性（SSCP）。HLA - B等位基因在非变性聚丙烯酰胺凝胶中显示出不同的电泳模式，这取决于第2外显子的核苷酸序列，表明SSCP分析可能是组织移植中HLA - B特异性的一种替代、有用且实用的HLA匹配系统。
