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Genotyping Parkinson disease-associated mitochondrial polymorphisms.
Jiang-Y; Ellis-T; Greenlee-AR
Clin Med Res 2004 May; 2(2):99-106
OBJECTIVE: The purpose of this study was to establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (T(m)) analysis. This technology should prove useful for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson disease (PD). METHODS: Mitochondrial DNA (mtDNA) was extracted from whole blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler (Roche Molecular Biochemical, Mannheim, Germany). Genotyping of 10 SNPs in 15 subjects was based on the analysis of allele-specific T(m) 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 nucleotide changes at positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, and 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, and 9.52 degrees C declines in T(m) of the detection probes, respectively. Genotyping of all 150 samples was verified by 100% correspondence with the results of sequencing. Fourteen subjects were haplogrouped by combining results for all 10 SNPs. CONCLUSION: A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes was 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.
Cell-biology; Cell-function; Cell-metabolism; Cell-morphology; Cellular-function; Cellular-structures
National Farm Medicine Center, Marshfield Clinic Research Foundation, Marshfield, Wisconsin 54449
Issue of Publication
Clinical Medicine & Research
Marshfield Clinic Research Foundation
Page last reviewed: September 2, 2020
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