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

Alexandros G. Georgakilas1, Stewart M. Holt2, Jessica M. Hair3, Charles W. Loftin3

1 Department of Physics, National Technical University of Athens, Zografou Campus, Athens, Greece, 2 Department of Physical Therapy Education, Elon University, Elon, North Carolina, 3 Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.11
DOI:  10.1002/0471143030.cb0611s49
Online Posting Date:  December, 2010
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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

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Treatment of Cells with an Oxidizing Agent and Preparation of Agarose Plugs
  • Basic Protocol 2: Running Single‐Cell Gel Electrophoresis (SCGE)
  • Support Protocol 1: Quantitative Analysis of SCGE Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Treatment of Cells with an Oxidizing Agent and Preparation of Agarose Plugs

  • 1% (w/v) molecular biology grade, normal‐melting‐point agarose in 1× PBS
  • Cells
  • Complete medium
  • Trypsin, optional
  • Phosphate buffered saline (PBS; appendix 2A)
  • Trypan blue
  • Hydrogen peroxide (H 2O 2)
  • 1 M Na 2EDTA
  • Low‐melting‐point agarose prepared in TE (molecular biology grade; Bio‐Rad)
  • Plug lysis solution (see recipe)
  • 1× TBE electrophoresis buffer (see recipe)
  • Appropriate repair enzymes and autoclaved and filtered buffers for enzymes (Fpg, EndoIII, EndoIV; New England Biolabs)
  • 1 M KCl, pH 7.8 ( appendix 2A), ice cold
  • Alkaline denaturation buffer (see recipe), ice cold
  • Autoclaved‐sterile microscope slides (3 × 5–in.) and 18‐mm2 coverslips (Corning)
  • 25‐cm2 cell culture flasks
  • Hemacytometer
  • 137Cs source
  • Refrigerated centrifuge
  • PFGE plug molds (BioRad)
  • 37°C incubation oven or thermal cycler

Basic Protocol 2: Running Single‐Cell Gel Electrophoresis (SCGE)

  • Agarose‐coated slides with treated DNA plugs (see protocol 1)
  • Neutral electrophoresis buffer (see recipe), ice cold
  • Alkaline (denaturation) electrophoresis running buffer (see recipe), ice cold
  • Neutralization buffer: 0.4 M Tris⋅Cl, pH 7.5 (store at 4°C)
  • 100% ethanol, ice cold
  • 30‐cm horizontal constant‐field gel electrophoresis chamber
  • Constant‐field agarose gel electrophoresis apparatus with power supply
  • Storage box for slides under dark condition

Support Protocol 1: Quantitative Analysis of SCGE Slides

  • SYBR Green DNA staining dye (Cambrex BioScience) diluted 1:10,000 (1×) in TE buffer (10,000× SybrGreen)
  • Fluorescence microscope (20×, 40×, or 60× objective lens) equipped with a monochrome CCD camera
  • Comet analysis software (e.g., CometScore, Tritek)
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Literature Cited

Literature Cited
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