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DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 291))

Abstract

This assay quantifies the amount of DNA double-strand break (DSB) damage in attached cell populations embedded in agarose and assayed for migratory DNA using pulsed-field gel electrophoresis (PFGE) with ethidium bromide staining. The assay can measure pre-existing 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 are 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 these repaired lanes in comparison with the total amount of DNA fragmentation following determination of a optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

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Authors and Affiliations

  1. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

    Nina Joshi & Stephen G. Grant

Authors
  1. Nina Joshi
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  2. Stephen G. Grant
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Editor information

Editors and Affiliations

  1. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

    Phouthone Keohavong  & Stephen G. Grant  & 

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© 2005 Humana Press Inc.

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Joshi, N., Grant, S.G. (2005). DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:121

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  • DOI: https://doi.org/10.1385/1-59259-840-4:121

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-084-7

  • Online ISBN: 978-1-59259-840-3

  • eBook Packages: Springer Protocols

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