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

Content Provider	Europe PMC
Author	Shoop, Wendy K. Gorsuch, Cassandra L. Bacman, Sandra R. Moraes, Carlos T.
Copyright Year	2022
Abstract	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.
ISSN	00219258
Volume Number	298
PubMed Central reference number	PMC9650046
Issue Number	11
PubMed reference number	36209825
Journal	The Journal of Biological Chemistry [J. Biol. Chem]
e-ISSN	1083351X
DOI	10.1016/j.jbc.2022.102574
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2022-10-06
Access Restriction	Open
Rights License	This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). © 2022 The Authors
Subject Keyword	mitochondrial DNA heteroplasmy mtDNA copy number digital PCR MELAS mitochondrial genetics Ct, cycle threshold dPCR, digital PCR mtDNA, mitochondrial DNA nDNA, nuclear DNA qPCR, quantitative PCR RFLP, restriction fragment-length polymorphism
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry