FALCON-qPCR: a new method for the quantification of oxidative lesions in mitochondrial DNA

Accurate quantification of oxidative mitochondrial DNA (mtDNA) lesions remains technically challengingdue to the limitations of existing assays, which often require large sample inputs, multi-day workflows,and offer limited sensitivity. Here we introduce FALCON-qPCR (Fpg-assisted Long-PCR), a streamlined,high-sensitivity method for quantifying oxidative damage in mtDNA. FALCON-qPCR couples digestionwith formamidopyrimidine [fapy]-DNA glycosylase (Fpg) to long-range PCR and qPCR-based normal-ization, enabling precise lesion quantification from as few as 10,000 cells (~300 ng total DNA) withina single day. The assay provides a robust dynamic range and reproducibility across diverse biologicalsystems, including human cell lines, hepatocellular carcinoma biopsies, and Caenorhabditis elegans.Compared with established methods, FALCON-qPCR exhibits markedly higher sensitivity in detectingmtDNA damage induced by hydrogen peroxide, antimycin A, and rotenone. Its performance was furtherdemonstrated in assessing mitochondrial toxicity of ruthenium-based compounds, highlighting its poten-tial for pharmacological screening. By integrating enzymatic lesion recognition with quantitative ampli-fication in a unified workflow, FALCON-qPCR eliminates the need for mitochondrial isolation. Thismethodological advance provides a rapid, accurate, and scalable platform for studying oxidative DNAdamage, with broad applicability in mitochondrial research and translational toxicology.