For genotyping KCNJ6 G-1250A, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and direct sequencing were adopted. To perform PCR-RFLP, the restriction enzyme BsmI (Toyobo Co., Ltd., Tokyo, Japan) and two primers of P5F and P6R 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 dioxyribonucleoside triphosphate (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 cycles of 95°C for 30 s, 60°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 65°C in a total of 15 µl reaction solution containing 10×M buffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 500 mM NaCl, 10 mM dithiothreitol), 0.3 U BsmI, and 3.5 µl PCR product as the substrate. The digestion products were analyzed by electrophoresis
