多药耐药基因C3435T与G2677T/A单核苷酸多态性检测方法的探讨

目的 建立简便、快速检测多药耐药基因(MDR1)C3435T与G2677T/A单核苷酸多态性(SNPs)的方法 .方法 针对MDR1 C3435T分别设计相对的两对引物-聚合酶链反应(PCR-CTPP)、序列特异性聚合酶链反应(PCR-SSP)及DNA测序方法 的引物,针对MDR1 G2677T/A分别设计PCR-SSP和DNA测序方法 的引物,优化PCR反应条件.将PCR-CTPP和PCR-SSP方法 的基因分型结果 与DNA测序结果 进行比对,确定准确性.在优化条件下,分别对50例健康体检者的外周血白细胞DNA进行MDR1 C3435T和C2677T/A基因型分析.结果 通过条件优化,PCR-CIPP、PCR-SSP方法 可快速的清晰区分MDR1 C3435T与G2677T/A的基因型,结果 与DNA测序方法 相符合.50例健康体检个体MDR1 C3435T与G2677T/A的基因型分布均符合Hardy-Weinberg平衡(P0.05).MDR1 C3435T PCR-CTPP结合G2677T/A PCR-SSP的检测方法 为最佳选择.结论PCR-CTPP、PCR-SSP方法 可简单、准确、经济、快速地检测MDR1 C3435T、G2677T/A SNPs,具有临床应用价值.

Study on methods for detection of multidrug-resistance genes MDR1 C3435T and G2677T/A single nucleotide polymorphisms

Objective To establish a convenient,fast method for detection of single nucleotide polymorphisms (SNP) of multidrug-resistance 1 gene (MDR1) C3435T and G2677T/A.Methods For MDR1 C3435T the primers of PCR-CTPP (polymerase chain reaction with confronting two-pair primers),PCR-SSP (sequence-specific primers) and DNA sequencing were designed,and the primers of PCR-SSP and DNA sequencing were designed for MDR1 G2677T/A.The conditions for PCR were optimized,and the amplified results were verified by DNA sequencing. The genotypes of 50 healthy cases were also detected by the above methods.Results The allele-specific bands were successfully amplified under the optimized conditions of both PCR-CTPP and PCR-SSP,and the genotypic results were consistent with those of DNA sequencing. The MDR1 C3435T and G2677T/A genotypic distributions were all accorded with Hardy-Weinberg equilibrium (P>0.05).The optimal combination was PCR-CTPP for MDR1 C3435T and PCR-SSP for G2677T/A.Conclusions PCR-CTPP and PCR-SSP are simple,accurate,rapid and economical methods for detection of SNP of MDR1 C3435T and G2677T/A,and can be applied in clinical research.