The enzyme-modified comet assay: enzyme incubation step in 2 vs 12-gels/slide systems

Publication date: Available online 22 November 2018Source: Mutation Research/Genetic Toxicology and Environmental MutagenesisAuthor(s): Damian Muruzabal, Sabine Langie, Bertrand Pourrut, Amaya AzquetaAbstractThe enzyme-modified comet assay is a commonly used method to detect specific DNA lesions. However, still a lot of errors are made by many users, leading to dubious results and even misinterpretations. This technical note describes some critical points in the use of the enzyme-modified comet assay, such as the enzyme concentration, the time of incubation, the format used and the equipment. To illustrate the importance of these conditions/parameters, titration experiments of formamidopyrimidine DNA glycosylase (Fpg) were performed using the 2 gels/slide and the 12 minigels/slide formats (plus the 12-Gel Comet Assay UnitTM). Incubation times of 15 and 30 min, and 1 h were used. Results showed that the 12 minigels/slide system requires a lower volume and concentration of Fpg. A longer time of incubation has a bigger impact when using such format.Moreover, the paper describes how to perform and interpret a titration experiment when using the enzyme-modified comet assay.
Source: Mutation Research Genetic Toxicology and Environmental Mutagenesis - Category: Genetics & Stem Cells Source Type: research