Unit

UNIT 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay)

  1. Alexandros G. Georgakilas1,2,
  2. Stewart M. Holt1,3,
  3. Jessica M. Hair1,
  4. Charles W. Loftin1

Published Online: 1 DEC 2010

DOI: 10.1002/0471143030.cb0611s49

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Georgakilas, A. G., Holt, S. M., Hair, J. M. and Loftin, C. W. 2010. Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay). Current Protocols in Cell Biology. 49:6.11:6.11.1–6.11.17.

Author Information

  1. 1

    Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina

  2. 2

    Department of Physics, National Technical University of Athens, Zografou Campus, Athens, Greece

  3. 3

    Department of Physical Therapy Education, Elon University, Elon, North Carolina

Publication History

  1. Published Online: 1 DEC 2010
  2. Published Print: DEC 2010

Abstract

The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results. Curr. Protoc. Cell Biol. 47:6.11.1-6.11.17. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • oxidatively-induced clustered DNA lesions;
  • single-cell gel electrophoresis;
  • repair enzymes;
  • complex DNA damage