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Figure 1.
MTHFR C677T gene polymorphism established by PCR- digestion by HinfI. The protocol of Frosst and coworkers was followed,6 with some modifications. The forward and reverse primers used were 5'-TGAAGGAGAAGGTGTCTGCGGGA-3' and 5'-AGGACGGTGCGGTGAGAGTG-3', respectively. The PCR conditions were 2 minutes at 94°C followed by 32 cycles (94°C for 30 seconds, 62°C for 30 seconds, 72°C for 60 seconds), and 5 minutes at 72°C after the last cycle. A 198 bp fragment was amplified by PCR and subjected to HinfI digestion (New England Biolabs, UK). The 677T allele contains an HinfI site resulting in 175 bp and 23 bp fragments, whereas a C at position 677 (677C) does not. The PCR and digestion products were analysed in 3% TBE agarose gels. Samples were categorised as homozygous for the thermolabile variant (677TT, lane 2), heterozygous for wild type and variant (C677T, lanes 1 and 3), or wild type (677CC, lane 4). Bl, negative control; M, 100 bp ladder molecular weight marker (Invitrogen, UK). The 23 bp fragments were not visible on agarose gels.