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.

Plain Language Summary

Mitochondria are tiny structures inside our cells that produce energy. When they work, they also create reactive molecules called ROS (reactive oxygen species). If ROS levels get too high, they can damage mitochondrial DNA (mtDNA), which may lead to diseases such as cancer and neurodegeneration. Measuring this damage is important, but current methods are slow, need large samples, and are not very sensitive. We developed a new method called FALCON-qPCR that measures mtDNA damage quickly and accurately. It uses a special enzyme to mark damaged DNA and then applies two types of PCR (a technique to copy DNA) to calculate how much damage is present. Unlike older methods, FALCON-qPCR works with very small samples – just 10,000 cells – and gives results in one day. We tested FALCON-qPCR on human cells, liver biopsies from cancer patients, and even tiny worms (C. elegans). In short, FALCON-qPCR is a fast, reliable, and versatile tool for studying mitochondrial DNA damage in research and clinical settings.