Quantitative assays for uracil-DNA glycosylase of high sensitivity |
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| Authors: | A R Morgan J Chlebek |
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| Affiliation: | Department of Biochemistry, University of Alberta, Edmonton, Canada. |
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| Abstract: | We have developed a sensitive fluorometric assay using bisulfite deaminated (C----U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30,000 Escherichia coli cells are needed for each point in a kinetic assay. |
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