HLA-D基因配型的单链构型多态性银染分析

ANALYSIS OF HLA-D GENES BY SINGLE STRAND CONFORMATION POLYMORPHISM WITH SILVER-STAINING

用PCR/单链构型多态性(SSCP)/银染方法做HLA-D基因配型。取供、受者DNA,对HLA-DQA、DRB、DQB及DPB位点作PCR(第2外显子多态部分),然后对产物进行SSCP,电泳条带经银染后分析结果,以确定供、受者HLA-D基因型是否一致,银染较之溴化乙锭染色灵敏度高25倍左右,为骨髓移植前进行HLA-DQA、DRB、DQB及DPB基因配型提供了较为灵敏的非放射性的手段。该方法亦可应用于其它单链构型多态性分析。

In this report, silver-staining was applied to PCR / SSCP for HLA-D matching before BMT. DNAs of both donor and recipient were obtained to perform PCR for HLA-DQA, DRB, DQB and DPB loci (polymorphic regions of the second exon). Then the products were subjected to SSCP. After silver-staining of the electrophoretic bands, the results were analysed to determine whether the HLA-D genotypes of donor and recipient were identical. It was shown that the sensitivity of silver-staining was 25 times higher than that of EB-staining and the SSCP developed by silver was a sensitive method for the HLA-D matching. This teachnique could also be used in other single strand conformation polymorphism analyses.