MTHFR Gene Genotyping from Blood DNA

Genomic DNA was extracted from peripheral blood samples using Nucleon BACC1 (GE Healthcare, Chalfont Saint Giles, UK) or QIAamp DNA Blood Mini (Qiagen, Hilden, Germany) kits, according to the manufacturer’s instructions. The DNA was titrated by a Qubit 2.0 fluorometer (Invitrogen, Singapore) using a Qubit dsDNA BR assay kit (Life Technologies, Carlsbad, CA, USA). The genotyping of the two SNPs in the MTHFR gene (including c.665C>T (rs1801133) and c.1298A>C (rs1801131)) was performed using pre-designed TaqMan 5′ exonuclease assays (Applied Biosystems, Foster City, CA, USA); subsequently, the fluorescence signals of the probes were detected by an ABI 7300 Real-Time PCR System (Applied Biosystems), according to the supplier’s protocol.