Development and optimization of a quantitative DNA base excision assay

The base excision repair (BER) system routinely removes damaged bases from DNA to conserve the integrity of the genome. The aims of this study were to design and optimize an assay using fluorescent kinetic data from molecular beacon (MB) oligonucleotides to generate an enzymatic model of base excision. Damaged bases, including 8-oxo-7,8-dihydroguanine (8oxoG) and uracil, were incorporated within 5’-fluorescein, 3’-Dabcyl labeled stem-loop oligonucleotides for use in this repair assay. Fluorescence data was recorded to monitor the kinetics of the base excision. Parameters were estimated using nonlinear regression to characterize the efficiency of repair proteins in whole cell extracts. This assay provides a simple, reproducible, and quantitative method to determine BER efficiency as a means of characterizing DNA repair capacity in cells.M.S., Biomedical Engineering -- Drexel University, 200