快速、高通量MTHFR基因C677T多态性检测方法的建立

A rapid and high throughput detection method of C677T polymorphism in MTHFR gene

目的:建立亚甲基四氢叶酸还原酶(Methylenetetrahydrofolatereductase,MTHFR)基因C677T多态性的快速检测方法.方法:提取健康人外周血基因组DNA,采用PCR扩增含677位点的MTHFR基因片段.对PCR产物中残余的dNTPs和游离单链引物消化处理后进行TDI反应,测定荧光偏振值并分析C677T基因型.同时采用PCRRFLP方法分析C677T基因型进行比较.结果:100例标本中,TDIFP分析MTHFRC677T基因型:C/C40例、T/T14例、C/T46例;PCRRFLP分析MTHFRC677T基因型:C/C40例、T/T12例、C/T48例;其中两种方法检测结果不一致的两例标本经焦磷酸微测序证实为T/T纯合子.结论:建立了一种新的基于TDIFP的MTHFRC677T基因分型方法,该方法较RFLP方法更为准确,且快速、简便、通量高,适用于临床大批量标本的筛查.

AIM: To develop a rapid and high throughput detection method of C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene. METHODS: Genomic DNA was isolated from human blood cells and the MTHFR gene fragment containing 677-site was amplified using PCR. The residual dNTPs and primers in the PCR products were removed by clean-up procedure. The template-directed dye-terminator incorporation (TDI) was performed and the MTHFR C677T genotypes were determined with fluorescence polarization assay. The MTHFR C677T genotypes were also analyzed by PCR-RFLP method. RESULTS: The MTHFR C677T genotypes were determined in 100 healthy Chinese using TDI-PF and PCR-RLFP methods, respectively. The results showed that the MTHFR C677T genotypes C/C, T/T and C/T were 40, 14 and 46 cases by TDI-FP method and 40, 12 and 48 by PCR-RLFP, respectively. The two inconsistent cases were further analyzed by pyrosequencing method and confirmed to be T/T genotype. CONCLUSION: A MTHFR C677T detection method has been developed based on template-directed dye-terminator incorporation with fluorescence polarization. This new method has higher accuracy than RFLP method for MTHFR genotyping. It is rapid, easy and high throughput and suitable for large-scale clinical screenings.