High Fidelity Restriction Endonucleases
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.
1 - 74 . (canceled)
75 . A method of identifying a fidelity index (FI) of a restriction endonuclease and one or more variants thereof;
comprising:
selecting a reaction buffer and a DNA substrate containing the binding and cleavage site of the restriction endonuclease;
permitting serially dilutions of the restriction endonuclease and the one or more variants thereof to cleave the DNA substrate; and
determining an FI for each of the restriction endonuclease and the one or more variants thereof.
76 . A method according to claim 75 , further comprising: comparing the FI for the restriction endonuclease to the F1 for a variant thereof to obtain an improvement factor for the variant.
77 . A method according to claim 76 , wherein the improvement factor is greater than 2.
78 . A method according to claim 75 , wherein the reaction buffer comprises potassium acetate, Tris acetate and magnesium acetate; or magnesium chloride.