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Updated: Apr 29, 2026
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection
Agnieszka Zylicz-Stachula1, Katarzyna Polska, Piotr Skowron
1Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk (Poland).
Abstract:
DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.
Insights
This study introduces a novel molecular tool to distinguish and quantify single (SSBs) and double (DSBs) DNA strand breaks. This method aids in assessing DNA damage and anticancer therapy efficacy.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA strand breaks (SBs) are critical cytotoxic DNA damage markers.
- Residual SBs correlate with cell death and anticancer therapy efficacy.
- Differentiating single (SSBs) and double (DSBs) strand breaks is crucial.
Purpose of the Study:
- To develop a molecular tool for differentiating and quantifying SSBs and DSBs.
- To enable precise assessment of DNA damage levels.
- To improve understanding of DNA damage in relation to therapeutic outcomes.
Main Methods:
- Utilized PCR amplification of a linear DNA fragment with a sensitizing nucleotide.
- Employed circularization and enzymatic supercoiling of the DNA fragment.
- Assayed SSB and DSB levels using gel electrophoresis after exposure to damaging factors.
Main Results:
- Successfully developed a method to differentiate between SSBs and DSBs.
- Quantitatively assessed DNA strand break levels.
- Applied the method to an artificial plasmid to assay photoinduced SBs.
Conclusions:
- The described molecular tool effectively distinguishes and quantifies SSBs and DSBs.
- This method provides a valuable approach for DNA damage assessment.
- The tool has potential applications in evaluating DNA damage and therapy response.
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