Simultaneous Detection of Multiple Polymorphisms in Cyp2b6 and Opmr1 by Nested Pcr: a Good Approach for Patients on Methadone

Objective: Cytochrome P450 2B6 (CYP2B6) is involved in the metabolism of several therapeutically important drugs and abused intoxicants including methadone. The preferential binding target for methadone is the μ opioid receptor OPRM1. Various SNPs in CYP2B6 and OPRM1 may contribute to clinical outcomes in methadone maintenance therapy. The aim of the present study was to develop a consistent and robust assay to identify such genotypic variants. Methods: We randomly selected patients in the Malay population who were addicted to heroin and had already commenced methadone therapy. We developed a two-step, multiplexed, nested PCR method to simultaneously identify 26 SNPs in CYP2B6 and OPRM1. Three amplicons covering seven regions of interest were amplified in the first PCR step. These amplicons were then used as templates for 11 sets of reactions in the second PCR step. Results: We successfully amplified DNA fragments containing 26 SNPs of interest from the DNA samples provided by patients receiving methadone therapy: C534T, G779A, G13076A, A454G, G24A, G820A, G942A, G31A, G438A, T106C, C440G, T397A, T15708C, T480C, C17T, G794A, A13072G, A76T, A62T, C64T, A136G, C15618T, G691C, G15631T, T802C, and A118G. Conclusion: The PCR-based method was sensitive and specific and is the first nested, allele-specific, multiplexed PCR method reported for the simultaneous detection of all 26 clinically important SNPs of CYP2B6 and OPMR1.