Zahari Zalina, Salleh Mohd Razali, Zahri Johari Mohd Khairi, Musa Nurfadhlina, Ismail Rusli
Pharmacogenetics Research Group, Institute for Research in Molecular Medicine, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
Malays J Med Sci. 2011 Oct;18(4):44-57.
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.
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.
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.
The method was found to be simple, rapid, specific, and reproducible for the simultaneous detection of the DRD2 polymorphisms.
多巴胺D2受体基因（DRD2）在许多疾病中发挥作用，如精神分裂症、帕金森病和成瘾行为。目前可用于检测DRD2多态性的方法成本高昂，且无法同时检测我们感兴趣的所有8种多态性（Val96Ala、Leu141Leu、Val154Ile、Pro310Ser、Ser311Cys、TaqI A、A - 241G和 - 141C Ins/Del）。因此，我们开发了一种巢式多重聚合酶链反应（PCR）用于同时检测这些多态性。
采用标准化方法从血液中提取基因组DNA。设计针对多态性位点3'端的特异性引物。开发了一种两步PCR方法。在第一次PCR中，特异性扩增DRD2基因外显子3至4、外显子7、启动子区域和3'区域。随后将产物用作第二次PCR的模板。进行测序以验证测试结果。
获得了与感兴趣的扩增产物相对应的特异性条带。该方法用于精神分裂症患者基因分型时具有可重复性和特异性。扩增序列与DRD2序列显示100%同源性。
该方法被发现对于同时检测DRD2多态性而言简单、快速、特异且可重复。
