Oligonucleotide mediated nucleic acid recombination
AssigneeCodexis Mayflower Holdings, LLC
InventorsAndreas Crameri, Willem P. C. Stemmer, Jeremy Minshull, Steven H. Bass, Mark Welch, John E. Ness, Claes Gustafsson, Phillip A. Patten
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Abstract
Methods of recombining nucleic acids, including homologous nucleic acids, are provided. Families of gene shuffling oligonucleotides and their use in recombination procedures, as well as polymerase and ligase mediated recombination methods are also provided.
Claims33
A method of recombining homologous nucleic acids, the method comprising:
- (i) hybridizing a chemically synthesized set of family gene shuffling oligonucleotides, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to at least one region of sequence diversity of the homologous nucleic acids;
- and,
- (ii) elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids.
A method of recombining homologous nucleic acids, the method comprising:
- (i) hybridizing a chemically synthesized set of family gene shuffling oligonucleotides, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to at least one region of sequence diversity of the homologous nucleic acids;
- (ii) elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids;
- denaturing the population of recombined nucleic acids, thereby providing denatured recombined nucleic acids;
- reannealing the denatured recombined nucleic acids;
- and
- extending or ligating the resulting reannealed recombined nucleic acids.
A method of recombining homologous nucleic acids, the method comprising:
- (i) hybridizing a chemically synthesized set of family gene shuffling oligonucleotides;
- and,
- (ii) elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids;
- and
- (iii) repeating (i) and (ii) using a new chemically synthesized set of family gene shuffling oligonucleotides derived from the population of recombined nucleic acids until the population of recombined nucleic acids reaches a desired diversity.
A method of recombining homologous nucleic acids, the method comprising:
- (i) hybridizing a chemically synthesized set of family gene shuffling oligonucleotides;
- and,
- (ii) elongating the set of family gene shuffling oligonucleotides, thereby providing a population of recombined nucleic acids;
- (iii) repeating (i) and (ii) using a new chemicall ynthesized set of family gene shuffling oligonucleotides derived from the population of recombined nucleic acids until the population of recombined nucleic acids reaches a desired diversity;
- denaturing the population of recombined nucleic acids, thereby providing denatured recombined nucleic acids;
- reannealing the denatured recombined nucleic acids;
- and
- extending or ligating the resulting reannealed recombined nucleic acids.
Dependencies are inferred from the claim text.