Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
AssigneeMaxygen, Inc.
InventorWillem P. C. Stemmer
Life of this patent
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Abstract
A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.
Claims42
A method of producing recombined polynucleotides with desired functional properties, the method comprising:
- (a) conducting multiple cycles of denaturation, renaturation and incubation in the presence of a polymerase on polynucleotide strands having sequences from a plurality of variants of a polynucleotide, the polynucleotide strands being partially annealed through regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of recombined polynucleotides in concatemeric form;
- (b) introducing the concatemers without monomerization into host cells;
- and,
- (c) selecting or screening the recombined polynucleotides for the desired functional property.
A method of producing a library of cells, comprising:
- recombining a population of nucleic acids in vitro to produce a set of nucleic acid variants;
- introducing the set of nucleic acid variants into a plurality of cells;
- and,
- culturing the cells under conditions whereby the nucleic acid variants recombine with themselves or with one or more additional nucleic acid in the cells, thereby producing a set of cells comprising resulting recombinant nucleic acids, the set constituting the library of cells.
Dependencies are inferred from the claim text.