The 10μl reaction mixture consists of 5μl of TaqMan® Genotyping Master Mix, 1μl of 40X Assay Mix (ABI) (8μM detection probe for each allele, 36μM forward and reverse primer each), 10 ng of genomic DNA diluted in 2ul of Tris EDTA (TE) pH 8.0 (Quality Biological, Inc; Gaithersburg, MD) and 2ul of PCR Water. Amplification was performed with a StepOne™ Real-Time PCR System v1.0 (ABI) using 48-well plates and the following amplification profile: 60°C for 30 s and 95°C for 10 min, followed by 50 cycles of 92°C for 15 s and 60°C for 1 min. After amplification, endpoint fluorescence intensity was measured directly in the reaction plates, directly on the ABI StepOne™ System. Genotypes were determined using a StepOne™ Software v1.0 (ABI). Four genotyping signal clusters were identified, representing asn40 and asp40 homozygotes, asn40/asp40 heterozygotes and DNAfree-template controls. Subject samples were handled the same as at least 3 controls of each of the above genotypes in each assay. The fluorescence intensity of the subject sample was compared to that of the control clusters and visually identified and classified as to correct genotype.
