Cromatografia

Journal of Chromatography B, 722 (1999) 225–254

Review

Single cell gel electrophoresis assay: methodology and applications
E. Rojas*, M.C. Lopez, M. Valverde
´ ´ ´ ´ Laboratorio de Genetica y Toxicologıa Molecular, Departamento de Genetica y Toxicologıa Ambiental, Instituto de Investigaciones Biomedicas, U.N. A.M., P.O. Box 70228, Ciudad Universitaria, CP 04510 Mexico D.F., MexicoAbstract The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studiespublished to date about the principles, the basic methodology with currently used variations. We also explore the applications of this assay in: genotoxicology, clinical area, DNA repair studies, environmental biomonitoring and human monitoring. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Reviews; Single cell gel electrophoresis; Comet assay; DNA

Contents
1. Introduction............................................................................................................................................................................ 2. The SCGE / Comet methodology............................................................................................................................................... 2.1. Lysis solution................................................................................................................................................................. 2.2. Unwinding and electrophoresis buffer .............................................................................................................................. 2.3. Other experimental variables........................................................................................................................................... 2.3.1. Cell suspension preparation.................................................................................................................................. 2.3.2. Slide preparation................................................................................................................................................. 2.3.3. Neutralization time .............................................................................................................................................. 2.3.4. Permanent slides ................................................................................................................................................. 2.3.5. DNA-specific dye andmagnification for data collection ......................................................................................... 2.3.6. Data analysis ...................................................................................................................................................... 2.3.7. Modifications for DNA repair studies and cross-linking agents............................................................................... 3. Applications of the assay ......................................................................................................................................................... 3.1. Studies on genotoxicity ...................................................................................................................................................3.2. Clinical applications........................................................................................................................................................ 3.3. DNA repair studies ......................................................................................................................................................... 3.4. Environmental biomonitoring...