Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
AssigneeCodexis, Inc.
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.
Claims1
A method for introducing one or more mutations into a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into double-stranded random fragments of a desired size, comprising:
- a) adding to the resultant population of double-stranded fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the template polynucleotide;
- b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;
- c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at regions of identity between the single-stranded fragments and formation of a mutagenized double-stranded polynucleotide;
- and
- d) repeating steps (b) and (c).
Dependencies are inferred from the claim text.