Genotyping of MTHFR C677T Polymorphism

To genotype the SNP of the MTHFR C677T (rs1801133) gene, Tetra-ARMS PCR was used. Three types of genotypes (C/C, C/T, and T/T) were found for the MTHFR C677T (rs1801133) gene. The base pair size used for Tetra ARMS PCR was 86 bp and 146 bp. For the amplification of the MTHFR C677T (rs1801133) gene, two forward primers and two reverse primers were used (Table 1). At the start of the reaction, two non-allele specific primers were amplified by the outer primers which produced outer fragments that served as a template for the attachment and elongation of inner primers.
A Thermocycler Master Cycler Gradient was used to perform PCR. PCR was carried out in a 20 µL container with 1 µL of DNA sample, 10 µL Master Mix, 0.1–0.5 mM of each primer, and 8 µL of DD-H₂O. The initial denaturation temperature was 95 °C for 5 min, further followed by 40 cycles of denaturation at 94 °C for 30 s. Annealing was performed at 52 °C for 75 s, followed by extension at 72 °C for 40 s. The final extension temperature was again 72 °C for about 7 min. After the completion of the PCR reaction, 20 μL of PCR resultant product was placed in the well with 2% agarose gel, dyed with ethidium bromide, soaked in TAE buffer, and let to run in an electric field. The gel was then examined under UV light using a gel documentation system (InGenius3, Syngene, UK).