Introduction: Genotyping of single nucleotide polymorphisms (SNPs) is a common method in genetic association studies. In these studies, numerous samples are required for genotyping to find an appropriate estimation for the association observed. Therefore, it is required to setup genotyping methods with a low cost and rapid speed. In this regard, PCR confronting two-pair primers (PCR-CTPP) is a cost-effective and time-saving method for SNP genotyping. In this study we optimized the PCR-CTPP for genotyping of methionine synthase-A2756G polymorphism by touchdown PCR. Methods: To PCR-CTPP purpose, the complete sequence of MTR gene was obtained from NCBI and location of A2756G polymorphism was detected on the sequence. Four specific primers (two outer and two inner primers) were designed on the A2756G location by Primer1 online web server, and then the melting temperature (Tm) of primers was optimized, manually. Finally four primers with equal Tm synthesized for further usages. For MTR A2756G genotyping, a polymerase chain reaction was performed by utilizing outers and inner primers. The touchdown PCR was used to optimize the amplification process. The SNP genotypes were analyzed by agarose gel electrophoresis. Finally, to insure the accuracy PCR-CTPP result some samples were applied to direct DNA sequencing. Results: Our observations showed that PCR-CTPP was optimized by touchdown PCR. After PCR-CTPP, we observed two bands (307bp and 192bp), two bands (307, 168 bp), and three bands (307bp, 192bp, 168bp) on agarose gel for AA, GG, and AG genotypes, respectively. In addition, DNA-direct sequencing confirmed the results of PCR-CTPP. Conclusion: Our findings suggest that the touchdown PCR is an efficient strategy to optimize the PCR-CTPP as a cost-effective and time-saving method for genotyping of MTR A2756G.
