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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

In a clinical setting, CYP2D6 metabolic status can be determined from the patients phenotype or genotype. An appropriate drug regimen that takes into account CYP2D6 activity can then be prescribed.1 For phenotyping, a CYP2D6-specific probe substrate is administered to a patient, and the ratio of unchanged drug to metabolite is measured by a method such as high-performance liquid chromatography or gas chromatography.4 Although this is the traditional method for determining metabolic status, it has several disadvantages, including lengthy assay times, intra-individual variability, and possible differences in the rate of metabolism of the probe substrate versus the actual drug.1 For the genotyping method, the SNPs are characterized at one or more loci within the CYP2D6. The SNP genotype provides an accurate and reliable prediction of CYP2D6 activity.5 The genotyping method is preferred because it has short assay times, has lower intra-individual variability, and requires a very small amount of patient sample for DNA isolation.1

In this Application Note, we use a two-color real-time qPCR protocol on the DNA Engine Opticon 2 fluorescence detection system to genotype one of the CYP2D6 SNPs that contains the *4 allele, in DNA from 211 patients. The *4 allele is a SNP at position 1934 (GenBank Accession #M33388), where a variant A nucleotide replaces the wild-type G nucleotide. Our assay was able to identify several individuals homozygous for the variant A allele who would be poor metabolizers of many clinical drugs.5


Materials and Methods
The Human CYP2D6*4 Allelic Discrimination Kit (4312555) and the TaqMan 2X Universal PC
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