Alkaline Comet Assay for Assessing DNA Damage in Individual Cells

Xinzhu Pu1, Zemin Wang2, James E. Klaunig2

1 Biomolecular Research Center, Boise State University, Boise, Idaho, 2 Department of Environmental Health, Indiana University, Bloomington, Indiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.12
DOI:  10.1002/0471140856.tx0312s65
Online Posting Date:  August, 2015
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Abstract

Single‐cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single‐cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double‐strand breaks, single‐strand breaks, alkali‐labile sites, DNA‐DNA/DNA‐protein cross‐linking, and incomplete excision repair sites. The inclusion of digestion of lesion‐specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. © 2015 by John Wiley & Sons, Inc.

Keywords: comet assay; DNA strand break; oxidative DNA damage; genotoxicity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Alkaline Comet Assay
  • Support Protocol 1: Preparation of Comet Assay Slide
  • Support Protocol 2: Sample Preparation for Comet Assay
  • Support Protocol 3: Storage of Whole Blood Sample from Human for Comet Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Alkaline Comet Assay

  Materials
  • 0.5% low‐melting‐point agarose (LMPA; e.g., Sigma; cat. no. A9414) in PBS ( appendix 2A)
  • Sample (i.e., cell suspension [see protocol 3] or cryopreserved white blood cells [see protocol 4])
  • Lysis buffer (see recipe)
  • Fpg reaction buffer (see recipe)
  • E. coli Fpg
  • Alkaline buffer/electrophoresis running buffer (see recipe)
  • Neutralization buffer (see recipe)
  • 70% ethanol
  • SYBR Gold Nucleic Acid Gel Stain (e.g., Life Technologies; cat. no. S‐11494)
  • Water bath with adjustable temperature
  • 1.5‐ml protein LoBind microcentrifuge tube (e.g., Eppendorf)
  • Precoated glass slides (see protocol 2)
  • Glass coverslips
  • 37°C incubator
  • Horizontal gel electrophoresis apparatus
  • Desiccant
  • Fluorescent microscope with fluorescein filter and image capturing device
  • Comet analysis software (e.g., Komet 7; Andor Technology)

Support Protocol 1: Preparation of Comet Assay Slide

  Materials
  • Methanol
  • 1.5% normal‐melting‐point agarose (e.g., HiMedia; cat. no. RM273)
  • Glass slides

Support Protocol 2: Sample Preparation for Comet Assay

  Materials
  • Cell culture or tissue sample
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Rubber policeman
  • 15‐ml conical centrifuge tube
  • Small dissecting scissors
  • 70‐μm nylon mesh cell strainer (e.g., Corning, Product #352350)
  • Hemocytometer

Support Protocol 3: Storage of Whole Blood Sample from Human for Comet Assay

  Materials
  • Isopropanol
  • Blood storage solution (see recipe)
  • Blood sample
  • Freezing container (e.g., Nalgene Mr. Frosty)
  • Sterile 2.0‐ml screw‐cap vial
  • EDTA‐coated blood collection tube
  • –80°C freezer
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Figures

Videos

Literature Cited

Literature Cited
  Azqueta, A. and Collins, A.R. 2013. The essential comet assay: A comprehensive guide to measuring DNA damage and repair. Arch. Toxicol. 87:949‐968.
  Azqueta, A., Gutzkow, K.B., Brunborg, G., and Collins, A.R. 2011a. Towards a more reliable comet assay: Optimising agarose concentration, unwinding time and electrophoresis conditions. Mutat. Res. 724:41‐45.
  Azqueta, A., Meier, S., Priestley, C., Gutzkow, K.B., Brunborg, G., Sallette, J., Soussaline, F., and Collins, A. 2011b. The influence of scoring method on variability in results obtained with the comet assay. Mutagenesis 26:393‐399.
  Brusick, D. 2001. Genetic toxicology. In Hayes’ Principles and Methods of Toxicology (A.W. Hayes, ed.) pp. 819‐858. CRC Press, Boca Raton, Fla.
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  Singh, N.P., McCoy, M.T., Tice, R.R., and Schneider, E.L. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175:184‐191.
  Sirota, N.P., Zhanataev, A.K., Kuznetsova, E.A., Khizhnyak, E.P., Anisina, E.A., and Durnev, A.D. 2014. Some causes of inter‐laboratory variation in the results of comet assay. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 770:16‐22.
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