Optimization of real-time PCR method to detect gene duplication of CYP2D6 / Noor Azrina Sanik

Cytochrome P450 2D6 is a polypeptide of 497 amino acids which extensively involved in metabolism of wide range of drugs such tamoxifen, chlorpheniramine, codeine and many more. The gene encodes for this enzyme resides on chromosome 22q13.1 near two neighboring pseudogenes; CYP2D7 and CYP2D8. In person carrying CYP2D6 gene duplication, this enzyme will be expressed more thus increase the toxicity of pro-drugs and reduce the effectiveness of active drugs. The purpose of this study is to optimize the real-time PCR assay in detection of the gene duplication of CYP2D6. Real-time PCR was used due to its advantages over traditional PCR. The DNA samples extracted from blood and the primers that have already been prepared were used. Temperature gradient was established using traditional PCR in order to obtain the optimal annealing temperature to be used in real-time PCR assay. The Ct values of samples were compared. Detection of gene duplication of CYP2D6 using traditional PCR was used to confirmed the presence of gene duplication of CYP2D6 in real-time PCR assay. It can be concluded that the DNA sample consisting gene duplication of CYP2D6 was successfully be detected using optimized real-time PCR assay. However, further study need to be done to obtain amplification reaction with greater efficiency and it should cover wider parameters of optimization. Once this method has been fully optimized, well designed and developed, the detection of gene duplication of CYP2D6 will be much easier. Patient response to the pharmacotherapy can be determined to improve the effectiveness as well as reducing the cost and duration of treatment.