Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells

Background: The "comet assay" has become an interesting and a very useful t ool for the analysis of the induction and amount of DNA damage in single ce lls thus offering the opportunity to measure the effectiveness of DNA repai r. On the basis of the Ostling and Johanson protocol we have developed a mo dified method with increased sensitivity and high reproducibility. Material and Methods: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. Results: In this presentation the influences of different methodological fa ctors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evalu ated. Additionally the authors will show a comparison of different fluoresc ent DNA dyes pointing out their advantages or disadvantages for "comet" ana lysis. The usefulness of this technique and its capabilities are exemplifie d by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. Conclusions: This paper presents data on the optimization and standardizati on of the original "comet assay" leading to an extremely fast and practicab le protocol in the field of single cell gel electrophoresis. After irradiat ion with 0.1 Gy an increase in the amount of DNA damage can be measured wit h high statistical significance and the DNA repair capacity of individual c ells after X-ray doses of 2 Gy can be analyzed with high reproducibility. T he results comparing DNA repair capacities of different donors point out th at the "comet assay may have the potential for the estimation of individual radiosensitivity.