Method for in vitro molecular evolution of protein function

A method for generating a polynucleotide sequence or population of sequences from at least one parent polynucleotide sequence encoding one or more protein motifs, comprising the steps of

• a) digesting the at least one parent polynucleotide sequence with at least one exonuclease to generate at least one population of fragments, wherein said digesting with at least one exonuclease comprises digesting a first parent polynucleotide sequence with a first exonuclease to produce a first population of fragments and digesting a second parent polynucleotide sequence with a second exonuclease to produce a second population of fragments;
• b) contacting said first and second population of fragments, wherein said fragments of said first and second population of fragments anneal;
• c) amplifying the annealed fragments of step b) to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotide
• wherein in step (a) at least one parameter of the reaction used for digestion of the first population of single stranded polynucleotide molecules is different from the equivalent parameter(s) used in the reaction for digestion of the second population of single stranded polynucleotide molecules.