( Surekha Karudapuram Ph...)Rapid SNP Genotyping of the Pharmacogenomically Important Allele CYP2D6*4 Using Real-Time qPCR on the DNA Engine Opticon 2 System

Surekha Karudapuram, Ph.D. and David Batey Ph.D.
MJ Research, Inc., South San Francisco, CA

Abstract
A two-color real-time quantitative PCR (qPCR) protocol was used on the MJ Research DNA Engine Opticon 2 system to genotype the CYP2D6*4 allele in 211 patient samples. Two hydrolysis probes, one labeled with VIC and the other labeled with FAM, were used to discriminate between the wild-type G nucleotide and the variant A nucleotide at position 1934 (*4 allele) of the CYP2D6 gene. An analysis feature within the Opticon Monitor software assigned one of the three possible genotypes to each sample. The results of this study highlight the ability of the Opticon 2 fluorescence detection system to accurately distinguish between the three SNP genotypes: GG homozygote, AA homozygote, and GA heterozygote.

Introduction
CYP2D6, or debrisoquine 4-hydroxylase, a member of the cytochrome P450 class of enzymes, is involved in the metabolism of clinically important drugs including cardiovascular agents and psychotropic drugs.¹ The human CYP2D6 gene contains numerous Single Nucleotide Polymorphisms (SNPs) that alter enzyme activity, with effects ranging from no activity to ultra rapid activity.^{2, 3} The different levels of enzyme activity result in individual differences in the ability to metabolize specific drugs. Based on these differences, individuals can be classified as poor metabolizers (PMs) who cannot metabolize CYP2D6-dependent drugs, extensive metabolizers (EMs) who metabolize CYP2D6 substrates normally, intermediate metabolizers (IMs) who have a metabolic rate between those of EMs and PMs, and ultrarapid metabolizers (UMs) who have a supernormal metabolic rate.^{1, 2}
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