The comet assay: high throughput use of FPG

Libro: Genotoxicity and DNA repair: a practical approach
Autores: Azqueta Oscoz, Amaya; Collins, A. R.
Editorial: Human Press, Springer
ISBN: 978-1-4939-1068-7
Página inicial-final: 199 - 217
Fecha de publicación: 2014
Resumen: The alkaline comet assay in its standard form is well established as a genotoxicity testing assay, widely used in screening novel chemicals and pharmaceuticals for potentially carcinogenic effects. Incorporation of a digestion of DNA with lesion-specific enzymes is an accepted modification which has allowed, for example, the quantitative assessment of levels of 8-oxoguanine in DNA as a measure of oxidative damage, using the enzyme formamidopyrimidine DNA glycosylase (FPG). However, FPG is not restricted to the measurement of oxidized bases, and we describe here its use in a wider context to detect various kinds of DNA damage. A limitation of the standard assay is the relatively low number of samples that can be run in one experiment (restricted by the number of microscope slides fitting in the electrophoresis tank). Recent developments of high throughput versions of the comet assay have alleviated this problem, and we describe a modification based on the use of 12 minigels on each slide. We provide a detailed protocol for running 12 minigels per slide, with the inclusion of FPG to obtain enhanced sensitivity. We emphasize the conditions of the comet assay that are most critical for reproducibility and accuracy.