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.
Claims69
A method for producing a polynucleotide having a desired functional property, said method comprising:
- (a) providing a population of homologous polynucleotides, wherein at least two of the related polynucleotides differ from each other by the presence of at least one deletion or insertion;
- (b) shuffling the homologous polynucleotides to produce a plurality of recombinant polynucleotides, and selecting or screening the population of recombinant polynucleotides to obtain at least one recombinant polynucleotide that has evolved toward the desired functional property;
- and,
- (c) shuffling the recombinant polynucleotide(s) produced in the previous step, and selecting or screening for at least one recombinant polynucleotide that has evolved toward the desired functional property.
A method for producing a polynueleotide having a desired property, said method comprising:
- conducting a multi-cycle polynucleotide extension process on overlapping segments having sequences of a population of related polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides, wherein said related polynucleotides differ from each other by the presence of deletions or insertions;
- and
- screening or selecting recombinant polynucleotides or expression products thereof to identify at least one recombinant polynucleotide having a desired functional property.
A method for producing a polynucleotide having a desired property, said method comprising:
- (a) choosing a plurality of polynucleotide variants to be shuffled, wherein at least one polynucleotide variant has been mutagenized;
- (b) conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands comprising sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the 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 recombinant polynucleotides;
- and
- (c) selecting and screening recombinant polynucleotides to identify at least one polynucleotide having a desired functional property.
A method of producing a polynucleotide having a desired functional property comprising:
- (a) providing a plurality of polynucleotides comprising exon sequences separated by an intron sequence, at least one of which polynucleotide is in cell-free form;
- (b) shuffling the population of polynucleotides to produce recombinant polynucleotides;
- (c) selecting or screening for recombinant polynucleotides that have evolved toward the desired property;
- (d) repeating steps 2) and 3) with the recombinant polynucleotides selected in step 3) until a recombinant polynucleotide has acquired the desired functional property.
Dependencies are inferred from the claim text.