at 37°C in a total of 10 µl reaction solution containing 10×NEBuffer 3 (500 mM Tris-HCl, pH 7.9, 100 mM MgCl2, 1000 mM NaCl, 10 mM dithiothreitol), 0.5 U BspEI, and 5 µl PCR product as the substrate. The digestion products were analyzed by electrophoresis using 1–2% agarose gel and ethidium bromide staining for visualization under ultraviolet illumination. A 65 bp digested DNA fragment is not easily distinguishable; therefore, the appearance of 395 bp, 332 bp, and both 395 bp and 332 bp DNA fragments corresponded to the A/A, G/G, and A/G genotypes, respectively, of the loaded sample. The failure rate of the RFLP genotyping assays was 3.571%. To perform the TaqMan allelic discrimination assay with a LightCycler 480 (Roche Diagnostics K.K., Tokyo, Japan), TaqMan® SNP Genotyping Assays (Life Technologies Japan Ltd.) containing sequence-specific forward and reverse primers to amplify the polymorphic sequence and two probes labeled with VIC® and FAM™ dye to detect both alleles of the KCNJ6 A1032G (Assay ID: C_15868122_10) were used. Real-time PCR was performed in a final volume of 10 µl containing 2×LightCycler® 480 Probes Master (Roche Diagnostics K.K.), 40×TaqMan® Gene Expression Assays, 5 ng genomic DNA as the template, and up to 10 µl
