Detection of restricted specificity of endonucleases by non-poisson analysis of their enzymatic digestion products

Endonucleases, enzymes of the hydrolase class that cleave DNA or RNA within the substrate molecule, play numerous critical roles in molecular biologic, pharmacologic, and pathologic events as well as constitute invaluable tools used in genetic analysis, gene cloning and DNA sequencing. These enzymes have two critical parameters, activity and site specificity. The former is easily measured in commonly used assays, but the latter must be analyzed by more complex, indirect assays that are often not even carried out. We now show how both parameters can be measured directly and simultaneously in the same assay, which is not significantly more labor intensive than the commonly used activity assay. The effectiveness and accuracy of this assay was tested on a series of five bacterial restriction enzymes which recognized, respectively, 1, 2, 3, 4, and a large number (26) of reactive sites on a small, uniform DNA substrate molecule. We found that the distributions of the reaction products of all five enzymes precisely followed that predicted by the Binominal and Poisson distributions for the number of sites recognized by each enzyme, and that these distributions could be used to determine this number experimentally. In this way a measure of site specificity can be made for each enzyme as well as its activity. This new assay should be used routinely in a number of laboratories that now employ conventional assays for endonuclease activity.