Development of a method based on alkaline gel electrophoresis for estimation of oxidative damage to DNA in Escherichia coli

Abstract

A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes. Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l-1 H2O2. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed. After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer. The lengths were correlated with survival of the cells exposed to H2O2 and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H2O2-caused DNA damage and repair in E. coli.