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

    XUE Li, BAI Yuª²Jie, ZHU Wenª²Wei, YANG Fang, YAN Xiaoª²Jun, DONG Xiuª²Zhen

    1Department of Medical Electronic Engineering, School of Biomedical Engineering, 2Institute of Genetic Diagnosis Technology of Chinese PLA, School of Pharmacy, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ 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 677ª²site was amplified using PCR. The residual dNTPs and primers in the PCR products were removed by cleanª²up procedure. The templateª²directed dyeª²terminator incorporation (TDI) was performed and the MTHFR C677T genotypes were determined with fluorescence polarization assay. The MTHFR C677T genotypes were also analyzed by PCRª²RFLP method. RESULTS: The MTHFR C677T genotypes were determined in 100 healthy Chinese using TDIª²PF and PCRª²RLFP methods, respectively. The results showed that the MTHFR C677T genotypes C/C, T/T and C/T were 40, 14 and 46 cases by TDIª²FP method and 40, 12 and 48 by PCRª²RLFP, 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 templateª²directed dyeª²terminator 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 largeª²scale clinical screenings. ......