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Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria
Published on: September 18, 2014
Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR
L Cavelier1, A Johannisson, U Gyllensten
1Department of Genetics and Pathology, University of Uppsala, Uppsala, S-751 85, Sweden.
Abstract:
Mitochondrial DNA (mtDNA) is a multicopy, maternally inherited, genome. Individuals frequently carry a mixture of genetically distinct mtDNA molecules whose proportions may vary between sexual generations or among tissues from the same individual. Analyses of the genetic composition of mitochondria have previously relied on electron microscopy and have not permitted the genotype of single mitochondria to be determined. We have developed flow cytometry techniques to isolate single mitochondrial particles and PCR-based assays to determine the mtDNA copy number and composition of individual particles. In a first application of this method, we studied mitochondrial particles from fibroblast cells heteroplasmic for the tRNA lys(8344) point mutation, associated with myoclonus epilepsy and ragged red fiber (MERRF). Individual mitochondrial particles contained between 0 and 11 mtDNA molecules with a mean of 2.0 (95% CI 1.6-2.4). The majority (75%) of the mitochondrial particles from which a PCR product was obtained contained only one type of mtDNA, consistent with the low mean mtDNA copy number. The method developed may be applied to studies of the copy number and distribution of mtDNA genomes in different cell types.
Insights
Researchers developed a new method to analyze mitochondrial DNA (mtDNA) composition in single mitochondria. This technique reveals that most individual mitochondria contain only one type of mtDNA, offering new insights into mitochondrial genetics.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Mitochondrial DNA (mtDNA) is a multicopy genome inherited maternally.
- Individuals often possess a mix of genetically distinct mtDNA molecules within cells and tissues.
- Previous methods like electron microscopy could not determine the genotype of single mitochondria.
Purpose of the Study:
- To develop novel flow cytometry and PCR-based techniques for analyzing single mitochondrial particles.
- To determine the mtDNA copy number and genetic composition within individual mitochondria.
- To investigate heteroplasmy in fibroblast cells carrying the tRNA lys(8344) point mutation linked to MERRF.
Main Methods:
- Utilized flow cytometry to isolate single mitochondrial particles.
- Employed PCR-based assays to quantify mtDNA copy number and identify genetic variants.
- Applied the method to fibroblast cells heteroplasmic for a specific mtDNA mutation associated with MERRF.
Main Results:
- Individual mitochondrial particles contained a mean of 2.0 mtDNA molecules (range 0-11).
- Approximately 75% of analyzed mitochondrial particles contained only one type of mtDNA.
- This suggests a low mean mtDNA copy number per mitochondrion and limited heteroplasmy at the single-particle level.
Conclusions:
- The developed method enables precise analysis of mtDNA copy number and composition in single mitochondria.
- Findings indicate a predominantly homoplasmic state for mtDNA within individual mitochondrial particles.
- This technique has broad applications for studying mtDNA distribution and heteroplasmy across various cell types.
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