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

Nature Protocols volume 18, pages 929–989 (2023)Cite this article

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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 14–16 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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Author notes

  1. These authors contributed equally: Andrew Collins, Peter Møller, Goran Gajski, Soňa Vodenková.

  2. These authors jointly supervised this work: Sabine A. S. Langie, Amaya Azqueta.

Authors and Affiliations

  1. Department of Nutrition, University of Oslo, Oslo, Norway

    Andrew Collins

  2. Department of Public Health, Section of Environmental Health, University of Copenhagen, Copenhagen, Denmark

    Peter Møller

  3. Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia

    Goran Gajski & Mirta Milić

  4. Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic

    Soňa Vodenková

  5. Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic

    Soňa Vodenková

  6. Oxidative Stress Group, Department of Environmental Health Sciences, Florida International University, Miami, FL, USA

    Abdulhadi Abdulwahed

  7. Biomedical Sciences Department, University of Bradford, Bradford, UK

    Diana Anderson

  8. Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

    Ezgi Eyluel Bankoglu & Helga Stopper

  9. Department of Human Sciences and Quality of Life Promotion, San Raffaele University, Rome, Italy

    Stefano Bonassi

  10. Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Roma, Rome, Italy

    Stefano Bonassi

  11. Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France

    Elisa Boutet-Robinet

  12. Division of Climate and Environmental Health, Norwegian Institute of Public Health, Oslo, Norway

    Gunnar Brunborg, Kristine B. Gutzkow & Ann-Karin Olsen

  13. Centre for Environmental Radioactivity (CoE CERAD 223268/50), Oslo, Norway

    Gunnar Brunborg, Kristine B. Gutzkow & Ann-Karin Olsen

  14. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Christy Chao

  15. Oxidative Stress Group, Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, USA

    Marcus S. Cooke

  16. Environmental Health Department, National Institute of Health, Porto, Portugal

    Carla Costa, Solange Costa & João Paulo Teixeira

  17. EPIUnit - Instituto de Saúde Pública, Universidade do Porto, Porto, Portugal

    Carla Costa, Solange Costa & João Paulo Teixeira

  18. Laboratory for Integrative and Translational Research in Population Health (ITR), Porto, Portugal

    Carla Costa, Solange Costa & João Paulo Teixeira

  19. Centre of BioMedical Research, SGPGIMS Campus, Lucknow, India

    Alok Dhawan

  20. Toxicology Department, AC MARCA Group, L’Hospitalet de Llobregat, Barcelona, Spain

    Joaquin de Lapuente

  21. Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Milan, Italy

    Cristian Del Bo’

  22. Aix-Marseille University, CEA, CNRS, Institute of Biosciences and Biotechnologies of Aix-Marseille, Saint-Paul-Lez-Durance, France

    Julien Dubus

  23. Health Effects Laboratory, Department of Environmental Chemistry, NILU-Norwegian Institute for Air Research, Kjeller, Norway

    Maria Dusinska, Naouale El Yamani & Elise Rundén-Pran

  24. School of Pharmacy and Life Sciences, The Robert Gordon University, Aberdeen, Scotland

    Susan J. Duthie

  25. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Bevin Engelward & Norah Owiti

  26. Genetics and Biotechnology Department and Veterinary and Animal Research Centre (CECAV), Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

    Isabel Gaivão

  27. Department NEUROFARBA, Section Pharmacology and Toxicology, University of Florence, Florence, Italy

    Lisa Giovannelli

  28. Department of Pharmacology & Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Maastricht, The Netherlands

    Roger Godschalk, Frederik-Jan van Schooten & Sabine A. S. Langie

  29. Centre for Environmental and Marine Studies (CESAM) and Department of Biology, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal

    Sofia Guilherme & Mário Pacheco

  30. School of Chemistry and Bioscience, Faculty of Life Sciences, Bradford University, Bradford, UK

    Khaled Habas

  31. Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Cerdanyola de Vallès, Spain

    Alba Hernández & Ricard Marcos

  32. Biology and Environmental Toxicology Group, Faculty of Science, Universidad Nacional de Educación a Distancia (UNED), Madrid, Spain

    Oscar Herrero

  33. Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy

    Marina Isidori

  34. School of Biological and Marine Sciences, University of Plymouth, Plymouth, UK

    Awadhesh N. Jha

  35. Institute of Cancer Research, Internal Medicine I, Medical University Vienna, Vienna, Austria

    Siegfried Knasmüller

  36. Department Circular Economy and Environment, the Netherlands Organisation for Applied Scientific Research-TNO, Utrecht, The Netherlands

    Ingeborg M. Kooter

  37. VITO-Health, VITO, Mol, Belgium

    Gudrun Koppen

  38. Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warsaw, Poland

    Marcin Kruszewski

  39. Department of Molecular Biology and Translational Research, Institute of Rural Health, Lublin, Poland

    Marcin Kruszewski

  40. H&TRC-Health & Technology Research Center, ESTeSL-Escola Superior de Tecnologia da Saúde, Instituto Politécnico de Lisboa, Lisbon, Portugal

    Carina Ladeira

  41. NOVA National School of Public Health, Public Health Research Centre, Universidade NOVA de Lisboa, Lisbon, Portugal

    Carina Ladeira

  42. Universidade da Coruña, Grupo DICOMOSA, CICA – Centro Interdisciplinar de Química e Bioloxía, Departamento de Psicología, Facultad de Ciencias de la Educación, A Coruña, Spain

    Blanca Laffon

  43. Instituto de Investigación Biomédica de A Coruña (INIBIC), A Coruña, Spain

    Blanca Laffon & Vanessa Valdiglesias

  44. Laboratory of Ecotoxicology, Faculty of Natural Sciences and Museum, National University of La Plata, La Plata, Argentina

    Marcelo Larramendy & Guillermo E. Liwszyc

  45. Anses, French Agency for Food, Environmental and Occupational Health and Safety, Fougeres Laboratory, Toxicology of Contaminants Unit, Fougères, France

    Ludovic Le Hégarat

  46. Department of Cardiothoracic Surgery, University of the Free State, Bloemfontein, South Africa

    Angélique Lewies

  47. Department of Biotechnology, University of Rzeszow, Rzeszow, Poland

    Anna Lewinska

  48. Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain

    Adela López de Cerain, Damian Muruzabal, Julen Sanz-Serrano & Amaya Azqueta

  49. IdiSNA, Navarra Institute for Health Research, Pamplona, Spain

    Adela López de Cerain & Amaya Azqueta

  50. Food and Drug Administration, National Center for Toxicological Research, Division of Genetic and Molecular Toxicology, Jefferson, AR, USA

    Mugimane Manjanatha

  51. Translational Biomedicine Laboratory, Graduate Program of Health Sciences, University of Southern Santa Catarina, Criciuma, Brazil

    Vanessa Moraes de Andrade

  52. Department of Pharmaceutical Sciences, Unit of Public Health, University of Perugia, Perugia, Italy

    Massimo Moretti

  53. Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

    Matjaž Novak & Bojana Žegura

  54. Department of Biology, CBMA-Centre of Molecular and Environmental Biology, University of Minho, Braga, Portugal

    Rui Oliveira

  55. Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research, Lucknow, India

    Alok K. Pandey

  56. Global Product Stewardship - Human Safety, The Procter & Gamble Co, Cincinnati, OH, USA

    Stefan Pfuhler

  57. Laboratoire Ecologie fonctionnelle et Environnement, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France

    Bertrand Pourrut

  58. Henkel AG & Co. KGaA, Düsseldorf, Germany

    Kerstin Reisinger

  59. Department of Genomic Medicine and Environmental Toxicology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, CU, Mexico City, Mexico

    Emilio Rojas & Mahara Valverde

  60. NorGenoTech AS, Oslo, Norway

    Sergey Shaposhnikov

  61. Norwegian Scientific Committee for Food and Environment, Oslo, Norway

    Ville Sipinen

  62. Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium

    Karen Smeets & Annelies Wouters

  63. Universidade da Coruña, Grupo NanoToxGen, CICA – Centro Interdisciplinar de Química e Bioloxía, Departamento de Biología, Facultad de Ciencias, A Coruña, Spain

    Vanessa Valdiglesias

  64. Triskelion BV, Zeist, The Netherlands

    Frederique van Acker

  65. Helix3 Inc., RTP, Morrisville, NC, USA

    Marie Vasquez

  66. FARMOVS, Bloemfontein, South Africa

    Johannes F. Wentzel

  67. Department of Biology, University of Rzeszow, Rzeszow, Poland

    Maciej Wnuk

  68. Biocev, 1st Medical Faculty, Charles University, Vestec, Czech Republic

    Tomas Zikmund

  69. Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, Munich, Germany

    Tomas Zikmund

Authors
  1. Andrew Collins
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  2. Peter Møller
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Contributions

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.

Corresponding author

Correspondence to Amaya Azqueta.

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The authors declare no competing interests.

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Nature Protocols thanks Vilena Kašuba and Bryant Nelson for their contribution to the peer review of this work.

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Related links

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

Supplementary information

Supplementary Information

Supplementary Protocols 1–13.

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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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