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

Accurate quantification of oxidative mitochondrial DNA (mtDNA) lesions remains technically challenging due 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 digestion with formamidopyrimidine [fapy]-DNA glycosylase (Fpg) to long-range PCR and qPCR-based normalization, enabling precise lesion quantification from as few as 10,000 cells (~300 ng total DNA) within a single day. The assay provides a robust dynamic range and reproducibility across diverse biological systems, including human cell lines, hepatocellular carcinoma biopsies, and Caenorhabditis elegans. Compared with established methods, FALCON-qPCR exhibits markedly higher sensitivity in detecting mtDNA damage induced by hydrogen peroxide, antimycin A, and rotenone. Its performance was further demonstrated in assessing mitochondrial toxicity of ruthenium-based compounds, highlighting its potential for pharmacological screening. By integrating enzymatic lesion recognition with quantitative amplification in a unified workflow, FALCON-qPCR eliminates the need for mitochondrial isolation. This methodological advance provides a rapid, accurate, and scalable platform for studying oxidative DNA damage, with broad applicability in mitochondrial research and translational toxicology.