A QUANTITATIVE MICROTITER PLATE NUCLEASE ASSAY BASED ON ETHIDIUM DNA FLUORESCENCE/

A microtiter plate assay was developed to quantitate the nuclease acti vity of the extracellular Serratia marcescens endonuclease under diffe rent buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video doc umentation system. Time courses of DNA cleavage were recorded and clea vage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nucl ease activity and can be carried out very quickly within a few minutes . It can also be used with RNA as substrate. With appropriate modifica tions it should be possible to adapt this assay for other enzymatic re actions which are accompanied by changes in absorbance or fluorescence . (C) 1996 Academic Press, Inc.