Patent ID: 7807408
Filing Date: 2010-10-05
Classification: C12N,C12Q

Abstract:
1. A method for producing an evolved polypeptide comprising: randomly fragmenting two or more double stranded parent nucleic acids and generating three or more nucleic acid fragments from each of the parent nucleic acids, wherein at least one of the parent nucleic acids is capable of encoding a protein, and at least one of the nucleic acid fragments encodes a fragment of the protein; ligating at least a subset of the nucleic acid fragments and generating shuffled nucleic acids, wherein the nucleic acid fragments and the shuffled nucleic acids are double stranded and at least one of the shuffled nucleic acids comprises nucleic acid fragments from at least two of the parent nucleic acids and encodes the evolved polypeptide; ligating at least one hairpin oligonucleotide to each end of at least a subset of the shuffled nucleic acids to cap each end of the subset of the shuffled nucleic acids, and forming capped shuffled nucleic acids thereby controlling the average length of the subset of the shuffled nucleic acids wherein each end of the capped shuffled nucleic acids can no longer ligate to other nucleic acid molecules, and at least one of the subset of the shuffled nucleic acids and at least one of the capped shuffled nucleic acids encode the evolved polypeptide; uncapping the capped shuffled nucleic acids by cleaving the hairpin oligonucleotide from the capped shuffled nucleic acids and generating uncapped shuffled nucleic acids encoding the evolved polypeptide; transforming the at least one of the uncapped shuffled nucleic acids encoding the evolved polypeptide into a host cell; and expressing the evolved polypeptide encoded by the at least one of the uncapped shuffled nucleic acids encoding the evolved polypeptide.