| 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 NruI, and the mutations are selected from G75, Q99, G155, or P22/R90. The mutation may be G75A, Q99A, G155A; or P22A and R90A. Also claimed are methods of identifying the FI of the aforementioned NruI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for NruI, permitting serially diluted NruI variants to cleave the DNA substrate, and determining the FI for the NruI variant. |
