摘要 |
Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer. The restriction endonuclease enzyme is NsiI, and the mutation is at F376. The mutation may be F376A. Also claimed are methods of identifying the FI of the aforementioned NsiI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for NsiI, permitting serially diluted NsiI variants to cleave the DNA substrate, and determining the FI for the NsiI variant. |