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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

ood samples. A total of 211 patient DNA samples were genotyped in duplicate, along with the GG and AA homozygous control DNA, GA heterozygous control DNA, and a no-template control. The data were then analyzed by plotting the C(t) value in channel 1 (FAM) against the C(t) value in channel 2 (VIC) for each sample, using the Opticon Monitor software. The data for a 96-well plate are shown in Figure 2. Four tightly clustered groups were generated. Group 1 (blue) contained samples homozygous for the G allele, group 2 (red) contained samples homozygous for the A allele, group 3 (green) contained samples that were GA heterozygotes and group 4 (orange) were no-template control (NTC) reactions.

The results of genotyping the patient samples are summarized in Table 3. Of the 211 samples, 153 patients (72.5%) were homozygous for the wild-type allele G, while 9 (4.3%) were homozygous for the variant allele A. The remaining 49 samples (23.2%) were heterozygotes. The patients with a heterozygous genotype containing one copy of the wild-type allele would be extensive metabolizers, while those with the homozygous AA genotype would be poor metabolizers. The genotypes were present at frequencies consistent with previous studies.5

The results of the real-time qPCR assay were confirmed by restriction enzyme digestion of the PCR product at a BstN1 site that spans the SNP site. For each of the 211 patient samples, the CYP2D6*4 genotype determined by the real-time qPCR assay was identical to that determined by restriction digestion (data not shown).


Discussion
In this study, we accurately genotyped 211 patient samples for the CYP2D6*4 allele using a 2-co
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