Optimization of Neutral Comet Assay for studying DNA double-strand breaks in pea and wheat

Abstract

This study describes an adaptation of the Comet assay under neutral conditions for mono- and dicotyledonous plants pea (Pisum sativum L.) and wheat (Triticum aestivum L.). Modifications concern lysis and electrophoresis steps, respectively. Electrophoresis was carried out varying the intensity of the electric field. A linear relationship between the percentages of DNA in the tail from control background with alteration of intensity was found. Trypan blue dye exclusion test was used in order to determine the intactness of nuclear membrane of the isolated nuclei from both plant model systems. Assessment was conducted on non-irradiated and irradiated nuclei on a monolayer with three doses of UVC. It was found that the share of intact nuclei (trypan blue negative ones) is about 95% in controls. Gradual dose-related increase of damaged nuclei was observed in both species, reaching statistical significance only at the higher dose applied.

Keywords

• neutral Comet assay
• trypan blue dye exclusion test
• UVC