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Detection of DNA Damage Induced by Topoisomerase II Inhibitors, Gamma Radiation and Crosslinking Agents Using the Comet Assay

  • Lori A. Hazlehurst
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 523)

Abstract

The comet assay is a simple gel electrophoresis method for visualizing and quantifying DNA damage. The comet assay is sensitive and reproducible and can be used to detect single-strand DNA breaks, double-strand DNA breaks, protein-associated DNA strand breaks and DNA crosslinks. The comet assay uses fluorescent DNA-binding dyes to detect both damaged DNA that resides in the tail region and undamaged DNA that is retained in the head region following gel electrophoresis. This assay is a single cell-based assay and thus is highly adaptable for measuring DNA damage in clinical samples. Furthermore, unlike other assays the detection of DNA damage is not dependent on the random incorporation of radiolabeled nucleotides. Again this can be problematic with clinical samples as proliferation rates are often slow and culturing of primary patient specimens for 48 h required to randomly label DNA is often not possible. In this chapter we will outline the comet assay for the detection of DNA damage induced by topoisomerase II inhibitors, cross-linking agents and gamma radiation.

Key words

Comet assay filter elution assay topoisomerase II poison DNA crosslinks DNA repair 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Lori A. Hazlehurst
    • 1
  1. 1.Department of Interdisciplinary Oncology, Experimental Therapeutics and Drug Discovery ProgramsH. Lee Moffitt Cancer Center and Research InstituteTampaUSA

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