Patent ID: 7943303

Claim:
A method comprising: (a) obtaining a library of plasmids, in which the individual plasmids comprise: (i) a gene coding for a wild-type restriction endonuclease that has been randomly mutagenized, and is positioned between a promoter operably linked to the gene and a known asymmetric recognition sequence for the wild-type restriction endonuclease; and (ii) a substrate site for nicking by a first nicking endonuclease adjacent to the asymmetric recognition sequence; (b) transforming a first population of host cells with the library of plasmids, the first population of host cells lacking a protective methylase for the wild type restriction endonuclease; (c) isolating the plasmids from the transformed first population of host cells, the isolated plasmids comprising supercoiled DNAs and open circular DNAs wherein the open circular DNAs contain a nick at the known asymmetric restriction sequence resulting from cleavage by a product of the randomly mutagenized restriction endonuclease gene having nicking activity, the open circular DNAs thus encoding a second nicking endonuclease gene; (d) combining the isolated plasmids in vitro with the first nicking endonuclease so as to permit nicking of the isolated plasmids at the substrate site such that the open circular DNAs with the nick at the known asymmetric restriction sequence are linearized; and (e) introducing the second nicking endonucleases gene into a second population of host cells premodified with a protective methylase and expressing the second nicking endonucleases gene.