Zazhytska M. Mechanisms of DNA track formation in single cell gel electrophoresis (comet assay)

Object: Spatial organization of DNA in the cell nucleus. Objective: To establish the physical mechanisms of DNA track (comet tail) formation during the comet electrophoresis of isolated cells and to identify factors that affect on the electrophoretic mobility of DNA. Methods: The isolation of lymphocytes from whole blood, lysis treatment of cells in agarose blocks, neutral and alkaline variants of comet assay, light and fluorescence microscopy, spectrophotometric methods, statistical and theoretical methods of chemical kinetics. Results: The kinetics of DNA track formation as well as external factors that affect it were investigated, an approach to assess the supercoiling level of DNA loop domains was proposed, the fact of reversibility of DNA exit was found out, an approach to differentiate different levels of DNA damages with intercalators was proposed, the model of DNA-matrix proteins interaction was confirmed. Novelty: the registration of DNA exit kinetics for intact and irradiated lymphocytes in neutral and alkaline electrophoresis was performed for the first time, also the reversibility of DNA exit was shown for the first time, DNA-nuclear matrix proteins interaction is essential for comet tail formation during electrophoresis. Scope: biophysics, biochemistry, molecular biology and genetics. Results of the thesis are introduced into the educational process in ESC "Institute of Biology", Taras Shevchenko National University, Kiev.