POLYMERASE CHAIN-REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS OF POLYMORPHISM IN DPA1 AND DPB1 GENES - A SIMPLE, ECONOMICAL, AND RAPID METHOD FOR HISTOCOMPATIBILITY TESTING
POLYMERASE CHAIN-REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS OF POLYMORPHISM IN DPA1 AND DPB1 GENES - A SIMPLE, ECONOMICAL, AND RAPID METHOD FOR HISTOCOMPATIBILITY TESTING
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DOI：
10.1016/0198-8859(92)90059-v
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发表时间：
1992-02-01
期刊：
影响因子：
2.7
通讯作者：
SASAZUKI, T
中科院分区：
文献类型：
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作者：
HOSHINO, S;KIMURA, A;SASAZUKI, T
根据单链DNA电泳迁移率的多样性（单链构象多态性，SSCP）进行了一项新的技术试验，以检测HLA-DP基因的多态性。对来自31个细胞系的基因组DNA进行聚合酶链反应（PCR）以扩增DPA 1或DPB 1基因的多态性外显子2，所述细胞系分别为2个和14个不同的DPA 1和DPB 1等位基因纯合的，以及来自正常个体的外周血细胞的另一DPB 1等位基因纯合的。在甲酰胺存在下通过加热使PCR样品变性以获得单链DNA，在中性聚丙烯酰胺凝胶中电泳，并通过银染色可视化。等位基因的差异检测的独特的电泳模式的每一条单链，这取决于序列特异性构象。15个DPB 1等位基因显示11种不同的电泳模式，留下4个等位基因组合没有区分。用另一对引物进行PCR扩增，并进行SSCP分析，可进一步区分这四种等位基因组合。在PCR中使用四对引物允许区分所有15个DPB 1等位基因测试。DPA 1基因第2外显子存在两个等位基因差异。此外，通过SSCP分析，还发现了DPA 1和DPB 1基因的新等位基因。PCR-SSCP分析方法简单、快速，不需要放射性物质，也不需要限制性酶，有望成为研究临床器官移植所需的精细HLA配型的有用工具。
A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.