Quantitative Analysis of DNA Double-Strand Breaks in Genomic DNA Using Standard Curve Method.

BACKGROUND: DNA double-strand breaks (DSBs) are the most lethal and dangerous type of lesions with significant implications for both cellular function and organismal health. The number of DSBs (NDSBs) across the genome reflects DNA damage severity. However, current quantification methods mainly rely on next-generation sequencing, which is laborious and expensive. This study aims to provide a simple, low-cost, and high-throughput standard curve-based method for quantifying genome-wide DSBs. METHOD: Genomic DNA from human, mouse, Arabidopsis, Saccharomyces cerevisiae, and Escherichia coli was digested by seven blunt-end restriction enzymes to generate DSB standards. Theoretical NDSBs for each standard were calculated based on restriction site frequency. Ligation-mediated quantitative PCR (LM-qPCR) was performed to obtain the Ct values, which were plotted against log-transformed NDSBs to construct standard curves. Method reliability was assessed by comparing results with neutral single-cell gel electrophoresis and γ-H2AX flow cytometry. RESULTS: All genomes were successfully digested by seven blunt-end restriction enzymes to produce standard DSB fragments. Standard curves demonstrated high linearity (R2 > 0.95), with intra- and inter-assay coefficients of variation of 1.101% and 2.528%, respectively. The detection limit was below 100 DSBs. Quantification results strongly correlated with traditional DSB detection methods (|r| > 0.9). CONCLUSION: This standard curve-based method enables accurate, reproducible quantification of genome-wide DSBs in various organisms. It is simple, low-cost, and easily standardized, offering a promising tool for applications in genotoxicity testing, environmental exposure monitoring, and DNA damage research.