Genotyping was performed with the TaqMan genotyping method.50 Briefly, the polymerase chain reactions (PCRs) were conducted with 384-well microplates. To ensure the quality of genotyping, negative control samples were included in each plate. The PCRs were performed with 5 ng of genomic DNA, 0.25 μL of TaqMan assay mix (Applied Biosystems, Inc, Foster City), and 2.5 μL of TaqMan universal PCR master mix in a total reaction volume of 5 μL. After activating the polymerase and denaturizing DNA by heating at 95°C for 10 minutes, 40 cycles at 92°C for 15 seconds and 60°C for 1 minute were performed. After the reaction, the fluorescence intensities of reporter 1 and 2 (reporter 1: VIC fluorescent dye, excitation,520[10] nm; emission,550[10] nm; reporter 2: FAM fluorescent dye, excitation,490[10] nm; emission,510[10] nm) were measured (data are given as central wavelength [bandwidth] of filters used to measure fluorescence) by the Analyst fluorescence plate reader (LJL Biosytems, Sunnyvale, California). Based on the ratio of fluorescence intensities, genotypes were scored by a euclidean clustering algorithm developed in our laboratory.51
