Single cell gel electrophoresis combined with lesion-specific enzymes to measure oxidative damage to DNA

Libro: Laboratory Methods in cell Biology: biochemistry and cell culture
Autores: Collins, A. R.; Azqueta Oscoz, Amaya
Editorial: Academic Press
Localidad: San Diego
ISBN: 978-0-12-405914-6
Página inicial-final: 69 - 92
Fecha de publicación: 2012
Resumen: The comet assay (single-cell gel electrophoresis) is a simple, sensitive method for measuring DNA strand breaks, widely used in genotoxicity testing, human biomonitoring, ecogenotoxicology and fundamental research into mechanisms of DNA damage and repair. Cells embedded in agarose on a glass slide are lysed, leaving supercoiled DNA loops attached to the nuclear matrix as "nucleoids". Electophoresis attracts DNA to the anode, but only those loops with breaks migrate, forming a comet-like image on fluorescence microscopy. The relative intensity of the comet tail reflects the frequency of DNA breaks, with a detection range up to a few thousand breaks per cell. DNA breaks, being produced by many diverse agents and as intermediates in DNA repair, are an unspecific marker of damage. More detailed information is obtained by incorporating a digestion with a lesion-specific endonuclease, after the lysis step. Here, we concentrate on the detection of strand breaks and oxidized bases in human peripheral blood mononuclear cells or cultured mammalian cells. We cover preparation of cells, precoating of slides, embedding the cells in agarose, lysis, enzyme digestion, alkaline electrophoresis, fixation and staining, and scoring of comets both visually and with computerized image analysis.