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An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Nature Protocols volume 15, pages 3844–3878 (2020)Cite this article

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Abstract

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids (‘naked’ supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract’s incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.

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Fig. 1: Schematic overview of the principle behind the sample extract incubation reaction and comet formation.
Fig. 2: Overview of the comet-based in vitro DNA repair assay. (using 2 gels/slide as example).
Fig. 3: Overview of an example assay setup for the comet-based in vitro DNA repair assay.
Fig. 4: Principle of optimization of the extract’s protein concentration.
Fig. 5: The 12-gels/slide format.
Fig. 6: Theoretical data illustrating how to select the optimal incubation time.
Fig. 7: Equipment setup for exposure of substrate cells.
Fig. 8: Overview of slides containing internal experimental controls and sample extract used to calculate the final DNA repair incision activity.
Fig. 9: Examples of results obtained with a BER assay.
Fig. 10: Examples of results obtained with a NER assay using UVC-treated substrate cells.
Fig. 11: Examples of results obtained with a NER assay using BPDE-treated substrate cells.

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Data availability

The authors declare that the majority of the data shown here as examples or anticipated results are available in the original papers. 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.A. thanks the Ministry of Economy, Industry and Competitiveness (‘Ramón y Cajal’ programme, RYC2013-14370) of the Spanish Government for personal support. P.V. acknowledges support from the National Science Foundation (19-10543S).

Author information

Author notes

  1. These authors contributed equally: Sona Vodenkova, Amaya Azqueta.

Authors and Affiliations

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

    Sona Vodenkova, Alena Opattova & Pavel Vodicka

  2. Department of Medical Genetics, Third Faculty of Medicine, Charles University, Prague, Czech Republic

    Sona Vodenkova

  3. Department of Pharmacology and Toxicology, University of Navarra, and IdiSNA, Navarra Institute for Health Research, Pamplona, Spain

    Amaya Azqueta

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

    Andrew Collins

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

    Maria Dusinska

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

    Isabel Gaivão

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

    Peter Møller

  8. Biomedical Center, Medical Faculty in Pilsen, Charles University, Prague, Czech Republic

    Alena Opattova & Pavel Vodicka

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

    Roger W. L. Godschalk & Sabine A. S. Langie

  10. Centre for Environmental Sciences, Hasselt University, Hasselt, Belgium

    Sabine A. S. Langie

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  1. Sona Vodenkova
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Contributions

S.V., A.A., R.W.L.G. and S.A.S.L. designed figures; S.A.S.L. provided anticipated results and managed the manuscript preparation; S.V., A.A. and S.A.S.L. drafted the paper; and A.C., M.D., P.M. A.O., I.G. and P.V. contributed to and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sabine A. S. Langie.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Ricard Marcos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Vodenkova, S., Azqueta, A., Collins, A. et al. An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity. Nat Protoc 15, 3844–3878 (2020). https://doi.org/10.1038/s41596-020-0401-x

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