A Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for the Detection of DRD2 Polymorphisms
- Author:
Zalina Zahari
;
Mohd Razali Salleh
;
Mohd Khairi Zahri @ Johari
;
Nurfadhlina Musa
;
Rusli Ismail
- Publication Type:Journal Article
- Keywords:
dopamine D2 receptor, genetics, genetic polymorphism, methods, nested PCR, reproducibility of results, specificity
- From:Malaysian Journal of Medical Sciences
2011;18(4):44-57
- CountryMalaysia
- Language:English
-
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.
- Full text:W020151023393277687847.pdf
