Table 2.
PCR-RFLP/Direct Sequencing Parameters for the Detection of 5 Candidate Loci.
| Locus | Forward Primer | Reverse Primer | PCR Program | Restriction Endonuclease/Direct Sequencing |
|---|---|---|---|---|
| MTHFR 677C>T | 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ | 5′-AGGACGGTGCGGTGAGAGTG-3′ | 95°C 5 min, 40× (95°C 30 s, 59°C 30 s, 72°C 30 s), 72°C 5 min | HinfI |
| MTHFR 1298A>C | 5′-AAGGAGGAGCTGCTGAAGATG-3′ | 5′-CTTTGCCATGTCCACAGCATG-3′ | 95°C 5 min, 40× (95°C 30 s, 51°C 30 s, 72°C 30 s), 72°C 5 min | MboII |
| MTR 2756A>G | 5′-TGTTCCCAGCTGTTAGATGAAAATC-3′ | 5′-GATCCAAAGCCTTTTACACTCCTC-3′ | 95°C 5 min, 40× (95°C 30 s, 52°C 30 s, 72°C 30 s), 72°C 5 min | HaeIII |
| MTRR 66A>G | 5′-CAGGCAAAGGCCATCGCAGAAGA-3′ | 5′-CACTTCCCAACCAAAATTCTTCAAAG-3′ | 95°C 5 min, 40× (95°C 30 s, 52°C 30 s, 72°C 30 s), 72°C 5 min | Direct sequencing |
| RFC-1 80G>A | 5′-AGTGTCACCTTCGTCCCCTC-3′ | 5′-CTCCCGCGTGAAGTT CTT-3′ | 95°C 5 min, 40× (95°C 30 s, 60°C 30 s, 72°C 30 s), 72°C 7 min | HhaI |
Abbreviations: MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; RFC-1, reduced folate carrier 1; PCR-RFLP, polymerase chain reaction–restriction fragment length polymorphism; min, minutes; s, seconds.