Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR.

Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.

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Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Conflict of interest Wendy Shoop and Cassandra Gorsuch are employees and conducted these experiments at Precision BioSciences. Sandra Bacman and Carlos T. Moraes collaborate with Precision BioSciences scientists.