Sequence detection systems software sds 2.3

Genomic DNA was extracted from anti-coagulated peripheral blood leukocytes by proteinase K digestion and phenol/chloroform extraction. The polymorphisms were genotyped using the TaqMan MGB technology (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The PCR reactions were carried out in a total volume of 5 μL containing TaqMan Universal Master Mix, 80X SNP Genotyping AssayMix, Dnase-free water and 10-ng genomic DNA. The PCR conditions were 50°C at 2 minutes, 95°C at 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The 384-well ABI 7900HT Real-Time PCR System (Applied Biosystems) was used for the genotyping assay, according to the manufacturer's instructions and the Sequence Detection Systems software (SDS 2.3; Applied Biosystems) was used to automatically collect and analyse the data and to generate the genotype calls. Four negative controls were included in each plate to ensure accuracy of the genotyping. Two people performed genotyping independently, in a blinded manner, to ensure quality control. Approximately 5% of the samples were randomly selected for repeated genotyping and the results were 100% concordant.