Genotyping was performed with the TaqMan genotyping method [27]. Briefly, the 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, CA, USA) 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 of 92°C for 15 seconds and 55°C for 1 minute were performed. After the reaction, the fluorescence intensities of reporter 1 and 2 (reporter 1:VIC, excitation = 520 ± 10 nm, emission = 550 ± 10 nm; reporter 2: FAM, excitation = 490 ± 10 nm, emission = 510 ± 10 nm) were measured by the Analyst fluorescence plate reader (LJL Biosytems, Sunnyvale, CA). Based on the ratio of fluorescence intensities, genotypes were scored by a Euclidean clustering algorithm developed in our laboratory [28].
