Work

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

Year: 2025

Type: article

Abstract: 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 ( R 2 > 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.

Source: Journal of Clinical Laboratory Analysis

DOI: 10.1002/jcla.70123

Authors: Lihuang Guo, Hanying Dai, Jiancheng Li, Chenwei Li, Yue Huang, Keqian Xu

Institutions: Central South University, Third Xiangya Hospital, Xiangya Hospital Central South University, Shenzhen Traditional Chinese Medicine Hospital

Language: English

Topic: DNA Repair Mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,120