摘要
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MDR1C3435T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MDR1G2677T/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MDR1C3435T和G2677T/A基因型分析。结果通过条件优化,PCR-CTPP、PCR-SSP方法可快速的清晰区分MDR1C3435T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MDR1C3435T与G2677T/A的基因型分布均符合Hardy-Weinberg平衡(P>0.05)。MDR1 C3435T PCR-CTPP结合G2677T/APCR-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MDR1C3435T、G2677T/ASNPs,具有临床应用价值。
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 HardyWeinberg 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.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2008年第6期444-447,共4页
Chinese Journal of Clinical Laboratory Science
关键词
多药耐药基因
多态性
单核苷酸
聚合酶链反应
DNA测序
muhidrug resistance-1
single nucleotide polymorphism
polymerase chain reaction
DNA sequencing
