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Updated: Aug 9, 2025
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Single Cell Analysis of Mitochondrial DNA Deletions
Helen A L Tuppen1, Amy K Reeve1, Amy E Vincent2
1Wellcome Centre for Mitochondrial Research, Translational and Clinical Research Institute, Faculty of Medical Sciences, Framlington Place, Newcastle University, Newcastle upon Tyne, UK.
Abstract:
Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.
Insights
Mitochondrial DNA deletions cause cellular dysfunction in aging and disease. New methods allow precise characterization of these deletions and their loads within single human cells for better understanding.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Mitochondrial DNA (mtDNA) deletions are implicated in human aging and disease, leading to mitochondrial dysfunction.
- The impact of mtDNA deletions depends on mutation load, deletion size, and breakpoint location, influencing oxidative phosphorylation complex deficiency.
- Mitochondrial dysfunction exhibits a mosaic pattern across tissues due to variations in deletion load and type between adjacent cells.
Purpose of the Study:
- To develop and present protocols for analyzing mtDNA deletions at the single-cell level.
- To enable characterization of deletion size, breakpoints, and mutation load in individual human cells.
Main Methods:
- Laser micro-dissection and single-cell lysis for tissue sample preparation.
- Long-range PCR for determining deletion size.
- mtDNA sequencing for breakpoint analysis.
- Real-time PCR for quantifying mutation load.
Main Results:
- Established protocols for comprehensive single-cell analysis of mtDNA deletions.
- Demonstrated ability to characterize deletion size, breakpoints, and mutation load from individual cells.
Conclusions:
- Accurate characterization of mtDNA deletions in single cells is crucial for understanding aging and disease.
- The developed methods provide essential tools for investigating the role of mtDNA deletions in cellular dysfunction.
