Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR
Open accessAbstract
Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.
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The inability to find changes in mtDNA copy number is inconsistent with recent studies demonstrating a reduction in mitochondrial number in diabetes and insulin resistance but similar to studies showing a decrease in OXPHOS gene expression.
“In some ways, our inability to find changes in mtDNA copy number are inconsistent with recent studies demonstrating a reduction in mitochondrial number in diabetes and insulin resistance (1,28) but similar to studies showing a decrease in OXPHOS gene expression (3,4).”
From A High-Fat Diet Coordinately Downregulates Genes Required for Mitochondrial Oxidative Phosphorylation in Skeletal Muscle (2005) · cites this paper as [28]
The Discussion reports no mtDNA depletion in muscle fibres from patients with multiple mtDNA deletions, but it does not address diabetes or insulin resistance. A search of our copy for those conditions and decreased OXPHOS gene expression found no passage supporting the studies described in the citing sentence. (LLM, conf=0.76)
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- Authors
- He, L.
- Affiliations
- Newcastle University (GB)
- Corresponding author
- He, L.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2002-07-15
- Volume
- 30
- Issue
- 14
- Pages
- 68e-68
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC135769
- OpenAlex
- W1992805337
- Cited by
- 320
- References
- 41
- Citations by year
- 2026: 7 · 2025: 5 · 2024: 16 · 2023: 3 · 2022: 11 · 2021: 12 · 2020: 15 · 2019: 11 · 2018: 17 · 2017: 14 · 2016: 13 · 2015: 14 · 2014: 23 · 2013: 12 · 2012: 11
- FWCI
- 3.94
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nucleic Acids Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Newcastle University ePrints (Newcastle Univesity)
- Funders
- Wellcome Trust
- Muscular Dystrophy UK
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- mitochondrial DNA deletions, heteroplasmy, single-cell analysis, mitochondrial disease, skeletal muscle fibers, mitochondrial genome sequencing
- MeSH terms
- DNA, Mitochondrial, Humans, Sensitivity and Specificity, Time Factors, Polymerase Chain Reaction, Mitochondrial Myopathies, Sequence Deletion, Muscle, Skeletal, Muscle Fibers, Skeletal
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