Factors influencing the sensitivity of the alkaline comet assay for detection of DNA damage after low doses of radiation

The alkaline comet assay is a sensitive technique for measuring single-strand breaks in DNA of individual mammalian cells. The method uses fluorescent microscopy to examine DNA patterns resulting from alkaline agarose gel electrophoresis of individual cells after staining with propidium iodide. Factors influencing the sensitivity of the assay to low doses of radiation were investigated in three parts. 1) A model was developed for the digital image supplied by the camera; and improvements in image quality were achieved by subtracting the dark-field image. 2) The conditions for staining of comets with propidium iodide were optimized for image quality by minimizing the background fluorescence through inclusion of 0.02 M NaCl, increasing in the staining time to several hours, and mimmizing the dye concentration. The dependence of comet fluorescence on dye concentration required a model of two binding modes with equilibrium constants, 3.7±0.6 x 10⁵ M⁻¹ and 3.6±0.2 x 10⁷ M⁻¹. These values are in general agreement with reported values for DNA in solution (3.0±0.2 x 10⁵ M⁻¹) and bound by nucleoprotein (1.1±0.4 x 10⁷ M⁻¹). A decline in the number of binding sites with increasing radiation damage explained the loss of comet fluorescence with increasing DNA damage. 3) Limitations in sensitivity due to background damage were investigated. A portion of background damage was found to be induced during cell suspension and preparation of agarose gels. The measurement of damage was not significantly influenced by alkaline lysis duration but was strongly affected by extended alkaline rinse duration. Enhanced sensitivity and minimum background damage were found using a cell suspension including culture medium, higher temperature agarose stock solution and a three hour rinse protocol. The sensitivity of the assay was found to be limited by additional background variations at an average level equivalent to 0.05 Gy. Sensitivity below this level would require averaging measurements from multiple, replicate samples.Science, Faculty ofPhysics and Astronomy, Department ofGraduat