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A Standardized Protocol for the In Vitro Comet-Based DNA Repair Assay

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Genotoxicity and DNA Repair

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

DNA repair is regarded as an important biomarker to be measured alongside DNA damage when considering the risk of cancer from environmental or genetic causes. Efficient repair deals with DNA lesions before they can disrupt replication and create mutations. Repair capacity can be readily assessed using an in vitro comet-based DNA repair assay, which is particularly useful in human biomonitoring studies where many samples are collected over an extended period, stored frozen, and analyzed at a later date. In this assay, a protein lysate is extracted from studied cells or tissues and is incubated with damage-containing substrate DNA. Repair proteins in extract are able to recognize and incise DNA lesions and cumulate DNA breaks, which are quantified with the comet assay. Here we provide detailed protocols for the in vitro estimation of base excision repair (on a substrate containing 8-oxoguanine induced by visible light in the presence of a photosensitizer) and nucleotide excision repair (with UV-induced pyrimidine dimers and 6-4 photoproducts as substrate). We describe the preparation of extracts from different kinds of source material (cultured cells, peripheral blood mononuclear cells, animal tissues, human biopsies) and emphasize the need for careful control of the extract concentration. Furthermore, we discuss not only conventional comet assay format (2 gels on microscope slide), but also a medium-throughput version (12 minigels in microscope slide), which is recommended for reduction of experimental variability.

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Acknowledgments

Ro 19-8022 was kindly provided by Hoffman la Roche. SL was supported by a postdoctoral grant from the AXA Research Fund. AA thanks the Ministerio de Educación y Ciencia (“Juan de la Cierva” programme, 2009) of the Spanish Government for personal support. IG thanks the Portuguese Science and Technology Foundation (FCT) under the Project PEst-OE/AGR/UI0772/2014.

Author information

Authors and Affiliations

  1. Department of Molecular Biology of Cancer, Institute of Experimental Medicine ASCR, Videnska 1083, Prague, Czech Republic

    Jana Slyskova

  2. Environmental Risk and Health Unit, Flemish Institute of Technological Research (VITO), Boeretang 200, Mol, 2400, Belgium

    Sabine A. S. Langie

  3. Department of Genetics and Biotechnology, CECAV, University of Trás-os-Montes and Alto Douro, Vila Real, 5000, Portugal

    Isabel Gaivão

  4. Department of Nutrition, University of Oslo, 1046, Blindern, Oslo, Norway

    Andrew R. Collins

  5. Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, Pamplona, 31009, Spain

    Amaya Azqueta

Authors
  1. Jana Slyskova
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  2. Sabine A. S. Langie
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  3. Isabel Gaivão
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  4. Andrew R. Collins
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  5. Amaya Azqueta
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Corresponding author

Correspondence to Jana Slyskova .

Editor information

Editors and Affiliations

  1. Universidad de Oviedo, Oviedo, Spain

    L. María Sierra

  2. Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

    Isabel Gaivão

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Slyskova, J., Langie, S.A.S., Gaivão, I., Collins, A.R., Azqueta, A. (2014). A Standardized Protocol for the In Vitro Comet-Based DNA Repair Assay. In: Sierra, L., Gaivão, I. (eds) Genotoxicity and DNA Repair. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1068-7_22

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  • DOI: https://doi.org/10.1007/978-1-4939-1068-7_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1067-0

  • Online ISBN: 978-1-4939-1068-7

  • eBook Packages: Springer Protocols

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