High fidelity restriction endonucleases

摘要

Inter alia, a composition characterized in that it comprises: a restriction endonuclease enzyme having at least one artificially introduced mutation and an overall fidelity index (FI) improvement factor of at least 2, the restriction endonuclease being capable of cleaving a substrate with at least a similar cleavage activity to that of the restriction endonuclease absent the artificially introduced mutation, in a predetermined buffer, wherein the artificially introduced mutation is the product of at least one of a targeted mutation, saturation mutagenesis, or a mutation introduced through a PCR amplification procedure, and wherein the restriction endonuclease absent the artificially introduced mutation is EcoRV and the artificially introduced mutation is selected from: D19A; E27A; and D19A/E27A is disclosed.