Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
AssigneeAffymax Technologies N.V.
InventorsWillem P. C. Stemmer, Andreas Crameri
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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.
Claims22
A method for evolving a green fluorescent protein (GFP) protein and a polynucleotide encoding same, comprising
- (1) providing a population of DNA segments encoding GFP at least one of which is in cell-free form;
- (2) shuffling the population of DNA segments encoding GFP whereby the segments recombine to form recombinant segments,
- (3) selecting or screening for recombinant segments having evolved toward a desired property;
- repeating (2) and (3) with the recombinant segments in (3) providing the population of DNA segments in (1) at each cycle of repetition until a recombinant segment has acquired the desired property.
A method for evolving a polynucleotide for acquisition of a desired property, comprising:
- (1) providing a population of variants of said polynucleotide, at least one of which is in cell-free form;
- (2) shuffling said variants of said polynucleotide to form recombinant polynucleotides;
- (3) selecting or screening for recombinant polynucleotides that have evolved toward the desired property;
- and
- (4) repeating steps (2) and (3) with the recombinant polynucleotides selected in step (3) until a recombinant polynucleotide has acquired the desired property.
Dependencies are inferred from the claim text.