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目的建立基于荧光杂交探针和熔解曲线分析方法,检测线粒体单核苷酸多态性的快速而可靠的技术,为与帕金森病相关的环境和遗传因素相互作用的人群流行病学研究提供技术支持。方法从全血中提取线粒体DNA,设计特异性引物和荧光素标记探针,在LightCycler仪器上进行实时PCR和熔解曲线分析,根据检测探针上特异等位基因的荧光探针的熔点温度(Tm值)给样本的10个单核苷酸多态性分型,测序验证熔解曲线分析结果。结果在线粒体DNA的1719、4580、7028、8251、9055、10398、12308、13368、13708和16391位点上的野生型向突变型变化,引起检测探针的Tm值依次降低6.51、8.29、3.26、7.82、4.79、2.84、2.73、9.04、8.53和9.52℃。熔解曲线分析获得的150个基因型分析结果与测序结果100/一致。结论荧光杂交探针技术能快速和有效地检测线粒体DNA多态性。这种方法将能够用于大规模的环境流行病学研究,并有助于研究帕金森病的发病机制,识别遗传易感生物标志和保护易感人群。
Objective To establish a rapid and reliable technique for detecting mitochondrial single nucleotide polymorphisms based on fluorescence hybridization probes and melting curve analysis and to provide techniques for epidemiological studies of populations that interact with environmental and genetic factors associated with Parkinson’s disease stand by. Methods Mitochondrial DNA was extracted from whole blood and specific primers and fluorescein-labeled probes were designed. Real-time PCR and melting curve analysis were performed on a LightCycler instrument. Based on the melting temperature of the fluorescent probe of the specific allele on the probe (Tm Value) to the sample of 10 single nucleotide polymorphisms typing, sequencing validation melting curve analysis results. As a result, the wild-type to the mutant at positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708 and 16391 of the mitochondrial DNA changed to cause the Tm value of the detection probe to decrease 6.51,8.29,3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53 and 9.52 ° C. The 150 genotype analysis results obtained from the melting curve analysis were consistent with the 100% of the sequencing results. Conclusion The fluorescent hybridization probe technique can detect mitochondrial DNA polymorphisms rapidly and effectively. This method will be able to be used in large-scale environmental epidemiological studies and to study the pathogenesis of Parkinson’s disease, identify genetic susceptible biomarkers and protect susceptible populations.