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Measuring DNA modifications with the comet assay: a compendium of protocols

Abstract

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

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Fig. 1: Overview of the standard and the enzyme-modified comet assay protocols.
Fig. 2: A schematic representation of interstrand crosslinks (ICLs) formation by cisplatin and detection with a variant of the alkaline comet assay.
Fig. 3: Representative images of three comets illustrating interstrand crosslinks (ICLs) detection following cisplatin treatment.
Fig. 4: Component parts of the 12-gel chamber unit.
Fig. 5: Images illustrating the 96-gel format using GelBond film.
Fig. 6: The CometChip platform.
Fig. 7: The vertical comet system.
Fig. 8: Principle of the DNA methylation-sensitive comet assay.
Fig. 9: Visualization of all comets and BrdU-positive comets only by fluorescence microscopy, using two filters.
Fig. 10: Example pictures of different types of signals seen in comet–FISH experiments after alkaline electrophoresis using U-2 OS cells.
Fig. 11: Overview of various species and different sample types that have been used in the comet assay.
Fig. 12: Titration steps in the enzyme-modified comet assay.
Fig. 13: Representative images of comets classified in five different classes for visual scoring.
Fig. 14: Detection of DNA crosslinks in a theoretical cell culture study.
Fig. 15: Assessment of DNA lesions by inhibition of late-stage excision repair processes in a theoretical cell culture study.
Fig. 16: Examples of data output of the enzyme-modified comet assay in theoretical samples.
Fig. 17: Levels of DNA migration in assay control samples from a biomonitoring study, encompassing 11 d of comet assay experiments.
Fig. 18: Example results from a study of Fpg-sensitive sites after exposure to diesel exhaust particles in cultured human HepG2 cells.

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Data availability

The majority of the data shown here as examples or anticipated results are available in the original papers. Figures 12 and 1416 are theoretical results, which are inspired by unpublished work from the authors’ laboratories. Other supporting data are available upon reasonable request to the corresponding author.

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Acknowledgements

We thank the hCOMET project (COST Action, CA 15132) for support. A. Azqueta thanks the Ministry of Science and Innovation (AGL2015-70640-R and PID2020-115348RB-I00) of the Spanish Government. S.G. thanks the national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia (IP, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July) for personal support. V.M.d.A. thanks the National Council of Technological and Scientific Development (CNPq—304203/2018-1) for personal support. D.M. thanks the program ‘Ayudas para la formación de profesorado universitario (FPU)’ of the Spanish Government for the predoctoral grant received. N.O. thanks the NIEHS Superfund Research Program ES ES027707 for personal support. J.S.-S. thanks the Government of Navarra for the predoctoral grant received. V.V. thanks the Ministerio de Educación, Cultura y Deporte (‘Beatriz Galindo’ program, BEAGAL18/00142) of the Spanish Government for personal support. M.S.C. acknowledges personal support from the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274-01. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health.

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P.M., S.V., S.A.S.L., K.B.G., M.S.C., B.E., J.F.W. and S.S. designed figures; P.M. provided anticipated results; A.C., G.G., P.M., S.V., S.A.S.L. and A. Azqueta drafted the paper and revised the manuscript; all other co-authors contributed to the Materials and Procedure sections; A.L.d.C., E.B.-R., F.J.v.S., M.S.C., S.C. and S.K. thoroughly reviewed the manuscript before submission; S.A.S.L. and A. Azqueta managed the manuscript preparation; all authors read and approved the final manuscript.

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Correspondence to Amaya Azqueta.

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Key references using this protocol

Gajski, G. et al. Mutat. Res. Rev. Mutat. Res. 779, 82–113 (2019): https://doi.org/10.1016/j.mrrev.2019.02.003

Gajski, G. et al. Mutat. Res. Rev. Mutat. Res. 781, 130–164 (2019): https://doi.org/10.1016/j.mrrev.2019.04.002

Azqueta, A. et al. Mutat. Res. Rev. Mutat. Res. 783, 108288 (2020): https://doi.org/10.1016/j.mrrev.2019.108288

Gajski, G. et al. Mutat. Res. Rev. Mutat. Res. 788, 108398 (2021): https://doi.org/10.1016/j.mrrev.2021.108398

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Collins, A., Møller, P., Gajski, G. et al. Measuring DNA modifications with the comet assay: a compendium of protocols. Nat Protoc 18, 929–989 (2023). https://doi.org/10.1038/s41596-022-00754-y

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