Design of a digital‐PCR assay to quantify fragmented human mitochondrial DNA

Digital PCR (dPCR) has been adapted to quantify the proportion of mitochondrial DNA (mtDNA) molecules without and with double-strand breaks (DSBs). This is based on a break-apart approach two differentially labeled target sequences distantly located in circular molecule. When targets amplify separated reaction partitions, original mtDNA molecule should be fragmented by DSBs at least, each different segment between targets. both same partition, it must correspond or linear These possibilities may distinguished through restriction endonuclease (RE) induced unique DSB within After RE-digestion, separation signals partitions indicate presence previous Otherwise, joint amplification partition would an initial that linearized endonuclease. The procedure was validated assaying proportions vitro digestion REs, evidencing perfect accordance expected theoretical values dPCR quantification. Samples from peripheral blood cells, cellular extracellular U2OS cell line, as well cells incubated ethidium bromide induce depletion, were evaluated. technique interest complement studies relation aging human disease, assess possible adverse effects certain drugs could related affectation mtDNA.