Development of a rapid electrophoretic assay for genomic DNA damage quantification.

Autor: Londero JEL; Post-Graduation Program in Biological Sciences: Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil., Schavinski CR; Post-Graduation Program in Biological Sciences: Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil., Silva FDD; Post-Graduation Program in Biological Sciences: Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil., Piccoli BC; Post-Graduation Program in Biological Sciences: Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil., Schuch AP; Post-Graduation Program in Biological Sciences: Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil. Electronic address: andre.schuch@ufsm.br.
Jazyk: angličtina
Zdroj: Ecotoxicology and environmental safety [Ecotoxicol Environ Saf] 2021 Mar 01; Vol. 210, pp. 111859. Date of Electronic Publication: 2021 Jan 08.
DOI: 10.1016/j.ecoenv.2020.111859
Abstrakt: Accuracy, sensitivity, simplicity, reproducibility, and low-cost are desirable requirements for genotoxicity assessment techniques. Here we describe a simple electrophoretic assay for genomic DNA lesions quantification (EAsy-GeL) based on subjecting DNA samples to rapid unwinding/renaturation treatments and neutral agarose gel electrophoresis. The experiments performed in this work involved different biological samples exposed to increasing environmental-simulated doses of ultraviolet-B (UVB) radiation, such as Escherichia coli, human leukocytes, and isolated human genomic DNA. DNA extraction was carried out using a universal and low-cost protocol, which takes about 4 h. Before electrophoresis migration, DNA samples were kept into a neutral buffer to detect double-strand breaks (DSBs) or subjected to a 5-min step of alkaline unwinding and neutral renaturation to detect single-strand breaks (SSBs) or incubated with the DNA repair enzyme T4-endonuclease V for the detection of cyclobutane pyrimidine dimers (CPDs) before the 5-min step of DNA unwinding/renaturation. Then, all DNA samples were separated by neutral agarose gel electrophoresis, the DNA average length of each lane was calculated through the use of free software, and the frequency of DNA breaks per kbp was determined by a simple rule of three. Dose-response experiments allowed the quantification of different levels of DNA damage per electrophoretic run, varying from a constant and low amount of DSBs/SSBs to high and dose-dependent levels of CPDs. Compared with other assays based on alkaline unwinding and gel electrophoresis, EAsy-GeL has important advantages for both environmental monitoring and laboratory testing purposes. The simplicity and applicability of this protocol to other types of DNA lesions, biological models, and agents make it ideal for genotoxicity, DNA repair studies, as well as for assessing exposure risks to ecosystems and human health.
(Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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