ABSTRACT
Objective To establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (Tm) curve analysis. This technique is suitable for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson's disease (PD). Methods mtDNA was extracted from the blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler. Genotyping of 10 SNPs was based on the analysis of allele-specific Tm of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. Results Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The changes at nucleotide positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, 9.52℃ decline in Tm of the detection probes respectively. Genotyping of all detected genes was verified by 100% correspondence with the results of sequencing. Conclusion A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes has ben developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.