DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay

The alkaline comet assay is used to detect DNA single-strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30-40% by low doses of ionizing radiation, N-methyl-N-nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells.