A standardized protocol for the in vitro comet-based DNA repair assay

Libro: Genotoxicity and DNA repair: a practical approach
Autores: Slyskova, J.; Langie, S. A. S.; Gaivao, I.; Collins, A. R.; Azqueta Oscoz, Amaya
Editorial: Human Press, Springer
ISBN: 978-1-4939-1068-7
Página inicial-final: 377 - 395
Fecha de publicación: 2014
Resumen: DNA repair is regarded as an important biomarker to be measured alongside DNA damage when considering the risk of cancer from environmental or genetic causes. Efficient repair deals with DNA lesions before they can disrupt replication and create mutations. Repair capacity can be readily assessed using an in vitro comet-based DNA repair assay, which is particularly useful in human biomonitoring studies where many samples are collected over an extended period, stored frozen, and analyzed at a later date. In this assay, a protein lysate is extracted from studied cells or tissues and is incubated with damage-containing substrate DNA. Repair proteins in extract are able to recognize and incise DNA lesions and cumulate DNA breaks, which are quantified with the comet assay. Here we provide detailed protocols for the in vitro estimation of base excision repair (on a substrate containing 8-oxoguanine induced by visible light in the presence of a photosensitizer) and nucleotide excision repair (with UV-induced pyrimidine dimers and 6-4 photoproducts as substrate). We describe the preparation of extracts from different kinds of source material (cultured cells, peripheral blood mononuclear cells, animal tissues, human biopsies) and emphasize the need for careful control of the extract concentration. Furthermore, we discuss not only conventional comet assay format (2 gels on microscope slide), but also a medium-throughput version (12 minigels in microscope slide), which is recommended for reduction of experimental variability.