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A Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for the Detection of DRD2 Polymorphisms
Zahari, Zalina; Salleh, Mohd Razali; Johari, Mohd Khairi Zahri; Musa, Nurfadhlina & Ismail, Rusli
Abstract
Background: The dopamine D2 receptor gene (DRD2) plays a role in many diseases such as schizophrenia, Parkinson’s disease, and addictive behaviour. Methods currently available for the detection of DRD2 polymorphisms are costly and cannot detect all 8 polymorphisms of our research interest simultaneously (Val96Ala, Leu141Leu, Val154Ile, Pro310Ser, Ser311Cys, TaqI A, A-241G, and −141C Ins/Del). Therefore, we developed a nested multiplex polymerase chain reaction (PCR) for simultaneous detection of these polymorphisms.
Methods: Genomic DNA was extracted from blood using standardised methods. Primers specific at the 3’-end for the polymorphic sites were designed. A two-step PCR method was developed. In the first PCR, a region from exon 3 to 4, exon 7, the promoter region, and the 3’-region of DRD2 were specifically amplified. The products were subsequently used as templates in the second PCR. Sequencing was performed to validate the test results.
Results: Specific bands corresponding to the amplified product of interest were obtained. The method was reproducible and specific when used to genotype patients with schizophrenia. The amplified sequences showed 100% homology to the DRD2 sequence.
Conclusion: The method was found to be simple, rapid, specific, and reproducible for the simultaneous detection of the DRD2 polymorphisms.
Keywords
dopamine D2 receptor, genetics, genetic polymorphism, methods, nested PCR, reproducibility of results, specificity
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