Related Experiment Video
Updated: Jun 5, 2026
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Detection of heteroplasmic mitochondrial DNA in single mitochondria
Joseph E Reiner1, Rani B Kishore, Barbara C Levin
1Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America. joseph.reiner@nist.gov
Background:
Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases.
Methodology:
This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion.
Significance:
Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.
Insights
Researchers developed a new method to analyze DNA within single mitochondria. This technique reveals heteroplasmy, or varying ratios of mutated mitochondrial DNA, within individual mitochondria, aiding the study of related diseases.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Mitochondrial DNA (mtDNA) mutations cause severe energy and respiratory disorders.
- Understanding mtDNA heteroplasmy distribution is crucial for disease development insights.
- Sub-cellular heterogeneity in mitochondria impacts disease pathogenesis.
Purpose of the Study:
- To develop a method for isolating and analyzing DNA from single mitochondria.
- To investigate the presence and distribution of heteroplasmy within individual mitochondria.
- To link mitochondrial DNA heterogeneity to the development of mitochondrial diseases.
Main Methods:
- Isolation of single mitochondria from lysed human HL-60 cells using optical tweezers.
- Micron-sized femtopipette for capturing individual mitochondria.
- Multi-round DNA amplification and sequencing for mtDNA analysis.
Main Results:
- Successful isolation and molecular analysis of mtDNA from single mitochondria.
- Detection of heteroplasmic mixtures within the mtDNA of individual mitochondria.
- Observed heteroplasmy ratios in single mitochondria align with those in single cells.
Conclusions:
- Individual mitochondria harbor heteroplasmic mtDNA at varying ratios.
- This method enables detailed analysis of mitochondrial DNA heterogeneity.
- Findings contribute to understanding the role of mtDNA distribution in disease.
