Detection of heteroplasmy in individual mitochondrial particles

Bobby G. Poe, Ciarán F. Duffy, Michael A. Greminger, Bradley J. Nelson, Edgar A. Arriaga

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration.

Original languageEnglish (US)
Pages (from-to)3397-3407
Number of pages11
JournalAnalytical and Bioanalytical Chemistry
Volume397
Issue number8
DOIs
StatePublished - Aug 2010

Bibliographical note

Funding Information:
Acknowledgements This work was supported by the National Institutes of Health (R03-AG18099 and R01-AG20866). B.G.P. acknowledges support from the NIH Chemistry/Biology Interface Training Grant (T32-GM08700).

Keywords

  • Deletion
  • Fluorescence
  • Heteroplasmy
  • Mitochondria
  • Mitochondrial DNA
  • Nucleoid
  • PCR

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