采用PCR-RFLP技术分析中国人HLA-DR5亚型

采用等位特异的限制性内切酶降解经PCR扩增的中国人HLA纯合细胞的DRB基因片段,根据相应的限制性片段长度多态性(PCR-RFLP),可十分快速而准确地从DNA水平区分DR5的两个亚型,并显示属于DRW12亚型的中国人HLA纯合细胞与参考细胞间电泳格局的差异,提示新的DRW12变异体的存在。该技术不必采用等位或顺序特异的寡核苷酸探针进行杂交,因而无需使用放射性同位素,展示了应用于HLA基因分型的良好前景。

Analysis of the Chinese HLA-DR5 Subtypes with PCR-RFLP Method

The second exons of HLA-DRB genes of Chinese homozygous cell lines were amplified with polymerase chain reaction (PCR), followed by digestion of the amplified DNA segments with the allele-specific restriction endonucleases Cfo 1 and Hinf I. The resulted patterns of restriction fragment length polymorphism (PCR-RF LP) in polyacrylamide gel electrophoresis were used for HLA-DR genotyping. With advantages such as short time-consuming, accuracy and no usage of radioisotope to label oligonucleotide probes for hybridization, the technique has been proved to be capable of subtyping five Chinese HLA-DR 5 cell lines to DRw11 and DRw12 when compared with the PCR-RFLP patterns of reference cell lines. In addition, three Chinese DRw12 cell lines Showed their differences with the reference DRw12 cell BM16 in the genotyping, suggesting that the Chinese DRw12 cell lines might be categorized as new DR5 subtypes or variants related to DRw12.