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The Comet Assay for DNA Damage and Repair: Principles, Applications, and Limitations
Abstract
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.
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“To evaluate whether Mdm2 expression would influence Nbs1-mediated DNA repair, we took advantage of the comet assay, which under neutral conditions measures double strand breaks on a per cell basis (41,42).”
From Mdm2 Binds to Nbs1 at Sites of DNA Damage and Regulates Double Strand Break Repair (2005) · cites this paper as [42]
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“Our results demonstrate that the toxicity of 5-FuCrNPs in CACO cells was dose-dependent and achieved better results than 5-Fu alone, as measured by the cellular reduction in MTT [62,63].”
From Novel Green Biosynthesis of 5-Fluorouracil Chromium Nanoparticles Using Harpullia pendula Extract for Treatment of Colorectal Cancer (2021) · cites this paper as [62]
Experimental — accuracy not yet measuredIn the citing paper
“Xia, et al. [64] showed that cells treated with chromium suffer from increased DNA damage, such as mutations, including double- and single-strand breaks at minute damage sites of DNA [62].”
From Novel Green Biosynthesis of 5-Fluorouracil Chromium Nanoparticles Using Harpullia pendula Extract for Treatment of Colorectal Cancer (2021) · cites this paper as [62]
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Metadata
- Authors
- Collins, A. R.
- Affiliations
- University of Oslo (NO)
- Corresponding author
- Collins, Andrew R.
- ORCID
- Collins, Andrew R.
- Venue
- Molecular Biotechnology
- Published
- 2004
- Volume
- 26
- Issue
- 3
- Pages
- 249-261
- ISSN
- 1073-6085 (ISSN-L)
- OpenAlex
- W2034485370
- Cited by
- 2,892
- References
- 46
- Citations by year
- 2026: 91 · 2025: 104 · 2024: 119 · 2023: 134 · 2022: 138 · 2021: 157 · 2020: 146 · 2019: 168 · 2018: 164 · 2017: 174 · 2016: 161 · 2015: 179 · 2014: 156 · 2013: 162 · 2012: 171
- FWCI
- 27.05
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Molecular Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Effects of Radiation Exposure
- Keywords
- comet assay, DNA strand breaks, DNA damage, DNA repair, genotoxicity testing, genotoxins, human biomonitoring, molecular epidemiology, DNA supercoiling
- MeSH terms
- Animals, DNA, DNA Damage, DNA Mutational Analysis, DNA Repair, Environmental Monitoring, Environmental Pollutants, Humans, Mutagens, Apoptosis, Molecular Epidemiology, Comet Assay
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