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Updated: Mar 6, 2026
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors
Artem P Gureev1, Ekaterina A Shaforostova1, Anatoly A Starkov2
1Department of Genetics, Cytology and Bioengineering, Voronezh State University, Voronezh, Russia.
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.
Insights
New PCR methods detect specific mitochondrial DNA (mtDNA) damage sites. This approach enhances genotoxicity testing for drugs and environmental toxins by identifying vulnerable mtDNA regions.
Area of Science:
- Mitochondrial biology
- Genotoxicology
- Molecular biology
Background:
- Mitochondrial DNA (mtDNA) damage is a key biomarker for genotoxicity.
- Current PCR methods amplify long mtDNA fragments, hindering precise damage site identification.
Purpose of the Study:
- To develop and validate a novel PCR method for precise detection of mtDNA damage sites.
- To identify mtDNA regions most susceptible to damage from toxins.
Main Methods:
- Developed primers to amplify ~2kb mouse mtDNA fragments, covering >95% of the genome.
- Enhanced mtDNA enrichment to prevent non-specific nuclear DNA amplification.
- Validated the method using rotenone and H2O2 in vivo and in vitro.
Main Results:
- Identified GTGR-sequence-enriched segments in the D-loop region as highly susceptible to damage.
- Demonstrated H2O2-induced mtDNA damage relaxes supercoiled conformation, increasing DNA polymerase accessibility.
- Observed a decrease in threshold cycle value correlating with H2O2-induced damage.
Conclusions:
- The modified PCR method offers simpler and more selective detection of mtDNA damage sites.
- This technique improves genotoxicity assessment of drugs and environmental toxins.
- Pinpoints specific mtDNA regions vulnerable to oxidative stress and chemical damage.
