All primers, probes and reagents were purchased from Applied Biosystems Inc. (ABI; Foster City, CA, USA). To ensure quality control, DNA samples from cases and controls were randomly distributed across a 384-well plate. SNPs were genotyped using TaqManTM fluorescence 5' exonuclease technology. Each 5 μl experiment contained 25 ng genomic DNA, 1.6X TaqMan assay primer/probe mix, 1X PCR buffer A, 2.5 mM MgCl2, 250 μM deoxyribonucleotide triphosphates (dNTPs) and 0.5 U AmpliTaq Gold polymerase. Thermocycling was performed as recommended by ABI using a Dual 384-Well GeneAmp® PCR System 9700 cycler and cycling conditions as follows: an initial denaturation stage at 95°C for 12 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for one minute, followed by storage at 4°C in the dark. Genotypes were determined on an ABI 7900HT Fast Real-Time PCR System using the allelic discrimination mode.
