For genotyping KCNJ6 A1032G, the PCR-RFLP method, TaqMan allelic discrimination assay (Life Technologies Japan Ltd.) and direct sequencing were adopted. To perform PCR-RFLP, the restriction enzyme BspEI (New England Biolabs, Inc., Ipswich, MA) was used. The forward primer P23F and the reverse primer P24R were used (Table 2). First, PCR was performed in a final volume of 10 µl containing 5×GoTaq™ reaction buffer (7.5 mM magnesium), 0.16 mM dNTP, 0.4 µM of each primer, 0.5 U GoTaq™ DNA polymerase (Promega K.K. Japan, Tokyo, Japan), and 5–50 ng extracted genomic DNA as the template. The PCR program was the following: 95°C for 2 min, followed by 35–40 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 8 min. The amplified DNA fragments were digested by the restriction enzyme 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
