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.
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.
Authors: D K Grandy; M A Marchionni; H Makam; R E Stofko; M Alfano; L Frothingham; J B Fischer; K J Burke-Howie; J R Bunzow; A C Server Journal: Proc Natl Acad Sci U S A Date: 1989-12 Impact factor: 11.205
Authors: E G Jönsson; M M Nöthen; F Grünhage; L Farde; Y Nakashima; P Propping; G C Sedvall Journal: Mol Psychiatry Date: 1999-05 Impact factor: 15.992
Authors: J Thompson; N Thomas; A Singleton; M Piggott; S Lloyd; E K Perry; C M Morris; R H Perry; I N Ferrier; J A Court Journal: Pharmacogenetics Date: 1997-12
Authors: Carolyn Sue Richards; Linda A Bradley; Jean Amos; Bernice Allitto; Wayne W Grody; Anne Maddalena; Matthew J McGinnis; Thomas W Prior; Bradley W Popovich; Michael S Watson; Glenn E Palomaki Journal: Genet Med Date: 2002 Sep-Oct Impact factor: 8.822