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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

PCR product following restriction digestion (data not shown). The recognition sequence of Bst NI [CC(A/T)GG] encompasses the site of the G to A allele at the third nucleotide.

Results
In this assay, two differentially labeled hydrolysis probes were used to discriminate between the wild-type G nucleotide and the variant A nucleotide at position 1934. The probe specific for the G nucleotide was labeled with VIC dye at the 5 end, while the probe specific for the A nucleotide contained a FAM label at the 5 end. DNA that is homozygous for the wild-type G nucleotide will be detected by the VIC-labeled probe and should generate only a VIC signal. DNA that is homozygous for the variant A nucleotide will be detected by the FAM probe and will only generate a FAM signal. DNA that is heterozygous will generate both a FAM and a VIC signal. The Opticon 2 system detects FAM fluorescence in channel 1 and VIC fluorescence in channel 2.

As a test of the assay, three control DNA templates were genotyped: a GG homozygote, an AA homozygote, and a GA heterozygote. As expected, reactions with the GG homozygote control DNA template showed only VIC fluorescence, the AA homozygote control DNA template showed only FAM fluorescence, and the GA heterozygote control DNA template showed both FAM and VIC fluorescence (Figure 1). The absence of carryover of FAM signal into channel 2 for the GG homozygote control template and VIC signal into channel 1 for the AA homozygote control template illustrates the excellent color-separation capability of the Opticon 2 system.

Having demonstrated the ability of the assay to distinguish the three genotypes, we next tested genomic DNA isolated from patient bl
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