Genotyping was performed by the 50-nuclease method using fluorogenic allele-specific probes. Oligonucleotide primer and probe sets were selected from the human TaqMan® Pre-Designed SNP Genotyping Assays or designed using Applied Biosystems’ Custom TaqMan® SNP Genotyping Assay service (ABI, Foster City, CA). PCR amplification reactions for Pre-Designed SNPs were performed in 5 μl volume containing 0.125μl 40X assay mix, 2.5 μl Master Mix, and 7.5 ng genomic DNA. PCR amplification reactions for Custom SNPs were performed in 5 μl volume containing 0.25μl 20X assay mix or 0.125μl 40X assay mix, 2.5μl Master Mix, and 7.5 ng genomic DNA. Reactions were incubated at 50°C for 2 min and at 95°C for 10 min, and amplified for 40 cycles at 92°C for 15 s and 60°C for 1 min. Allele-specific signals were distinguished by measuring endpoint 6-FAM or VIC fluorescence intensities using an ABI 7900HT Sequence Detection System and genotypes were generated using SDS v2.1 software (Applied Biosystems). As a quality control, 20% of genotypes were repeated. PCR amplification failed or provided ambiguous genotype results in 2.8% of SNP Markers.
