title: Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors.
abstract: Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (∼8-10kb), which complicates detecting exact sites of mtDNA damage. To identify the mtDNA regions most susceptible to damage, we have developed and validated a set of primers to amplify ∼2kb long fragments, while covering over 95% of mouse mtDNA. We have modified the detection method by greatly increasing the enrichment of mtDNA, which allows us solving the problem of non-specific primer annealing to nuclear DNA. To validate our approach, we have determined the most damage-susceptible mtDNA regions in mice treated in vivo and in vitro with rotenone and H2O2. The GTGR-sequence-enriched mtDNA segments located in the D-loop region were found to be especially susceptible to damage. Further, we demonstrate that H2O2-induced mtDNA damage facilitates the relaxation of mtDNA supercoiled conformation, making the sequences with minimal damage more accessible to DNA polymerase, which, in turn, results in a decrease in threshold cycle value. Overall, our modified PCR method is simpler and more selective to the specific sites of damage in mtDNA.
authors.affiliation: Department of Genetics, Cytology and Bioengineering, Voronezh State University, Voronezh, Russia.
authors.forename: Artem P
authors.initials: AP
authors.lastname: Gureev
authors.affiliation: Department of Genetics, Cytology and Bioengineering, Voronezh State University, Voronezh, Russia.
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Shaforostova
authors.affiliation: Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, NY, USA. Electronic address: ans2024@med.cornell.edu.
authors.forename: Anatoly A
authors.initials: AA
authors.lastname: Starkov
authors.affiliation: Department of Genetics, Cytology and Bioengineering, Voronezh State University, Voronezh, Russia.
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Popov
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000633:toxicity; D008099:Liver / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012402:Rotenone / Q000633:toxicity
chemical_list: D004272:DNA, Mitochondrial; D012402:Rotenone; D006861:Hydrogen Peroxide
keywords: Hydrogen peroxide; Mitochondrial DNA; Oxidative damage; RTGR-sequence; Rotenone