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Rapid SNP Genotyping of the Pharmacogenomically Important Allele CYP2D6*4 Using Real-Time qPCR on the DNA Engine Opticon 2 System

R Master Mix (4304437) were obtained from Applied Biosystems. The genotypes of the following DNA templates were analyzed: Control DNA for Allele 1 (G) and Allele 2 (A), a mixed control DNA that contained equal amounts of Allele 1 and 2, and 211 human DNA samples. DNA was isolated from patient blood samples by MaximBio (So. San Francisco, CA) using their genomic DNA isolation kit (EXT-0001). Reaction components were assembled in Hard-Shell microplates with white wells and white skirts (MJ Research, HSP-9655), and sealed with ultra-clear strip caps (MJ Research, TCS-0803). Volumes of individual components are shown in Table 1.

Following reaction assembly, the microplates were transferred to the DNA Engine Opticon 2 real-time system (MJ Research) and thermal cycling was performed in calculated mode according to the program listed in Table 2.

The fluorescence-threshold line was set as described in the DNA Engine Opticon 2 System Operation Manual. Briefly, the threshold line was set manually such that the line intersected the fluorescence traces at the point where signals surpassed background noise and began to increase. The threshold line was used to determine cycle threshold (C(t)) values for each well. The same threshold was applied to all wells, thereby ensuring accurate comparison of samples and controls.

To validate the results of the real-time qPCR protocol, a 613bp region spanning the SNP site was amplified from each patients sample DNA using primers with the sequence: forward 5-AGA-GAC-GAG-GTGCCC- C-3 and reverse 5-AAA-TCC-TGC-TCT-TCC-GAG-3. The presence of the wild-type (G) nucleotide was then determined by assaying for the absence of a Bst NI site in the
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