Development of Primer-Special TaqMan® PCR

Background: CYP2C9 *3 (1075A/C) is an inherited single nuclear polymorphism (SNP) of cytochrome P450 (CYP) 2C9, which affects the activity of the enzyme. In vitro studies with several drugs have indicated that the CYP2C9 *3 variant has an impaired capacity for drug metabolism. Therefore an efficient detection assay for this mutation may be important for clinical dose adjustment. Objective: The aim of this work was to develop an appropriate tool for detection of the CYP2C9 *3 polymorphism in the clinical laboratory. Study Design: The previously described TaqMan® mismatch amplification mutation assay (TaqMAMA) was modified to a primer-special (PS)-TaqMan® PCR to satisfy the high-throughput requirements of a clinical laboratory. 404 genomic DNA samples from South Chinese individuals were genotyped to test the detection system. The results were checked by bi-directional sequencing. Results: PS-TaqMan® PCR could correctly genotype the CYP2C9 allele from a genomic template at a concentration of 1 × 10 4 to 1 × 10 11 copies/PCR. Among the 404 genomic DNA samples, 24 heterozygotes and 380 wild-type homozygotes were detected and confirmed by bi-directional sequencing. Conclusion: PS-TaqMan® PCR was successfully developed for CYP2C9 *3 detection. This efficient, reliable, high-throughput tool could satisfy the requirements of a clinical laboratory test.