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

Abstract

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 (N_DSBs) 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 N_DSBs 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 N_DSBs 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.