Comet assay

No international standard exists

Principle

Genotoxicity test with eukaryote cells on the base of DNA damage

Abstract

The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in eukaryotic cells. The test design can be performed with almost every kind of cell typ and is independent from the cell cycle. The DNA damage can be proven in proliferating as well as in non-proliferating cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software.

Criteria

A positive effect shows a significant increase of DNA fragmentation. The statistical verification of the significance can be performed with statistic tests as Kruskall-Wallis or H-Test.

Test substance properties

The Comet Assay is applicable for solid, soluble substances as well as for liquids and water samples.