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Pulsed-Field Gel Electrophoresis Analysis of Multicellular DNA Double-Strand Break Damage and Repair

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

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Abstract

This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

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Author information

Authors and Affiliations

  1. Public Health Program, Nova Southeastern University, Fort Lauderdale, FL, 33314-7796, USA

    Stephen G. Grant

  2. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA

    Nina Joshi

Authors
  1. Nina Joshi
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  2. Stephen G. Grant
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Corresponding author

Correspondence to Stephen G. Grant .

Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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© 2014 Humana Press

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Joshi, N., Grant, S.G. (2014). Pulsed-Field Gel Electrophoresis Analysis of Multicellular DNA Double-Strand Break Damage and Repair. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_16

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

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