Oligonucleotide mediated nucleic acid recombination

• US 8,058,001 B2
• Filed: 09/11/2009
• Issued: 11/15/2011
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holdings, LLC (Codexis, Inc.)
• Inventors: Crameri, Andreas, Ness, John E., Patten, Phillip A., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C., Bass, Steven H., Welch, Mark
• Primary Examiner(s): Whisenant; Ethan C
• Application Number:
  US12/557,829
• Publication Number:
  US 20100184627A1
• Time in Patent Office: 795 Days
• Field of Search: 435/6, 435/440, 536/22.1, 536/23.1, 536/25.3
• US Class Current: 435/6
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

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.

Claims

1. 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.

2. The method of claim 1, wherein the population of recombined nucleic acids has greater molecular diversity than the homologous nucleic acids.

3. The method of claim 1, wherein at least one nucleic acid in the population of recombined nucleic acids includes regions of diversity from multiple homologous nucleic acids.

4. The method of claim 1, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids based on diversity of the population of recombined nucleic acids.

5. The method of claim 1, further comprising 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.

6. The method of claim 5, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property prior to repeating (i) and (ii).

7. The method of claim 1, wherein the chemically synthesized set of family gene shuffling oligonucleotides comprises a set of crossover family diversity oligonucleotides.

8. The method of claim 1, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to multiple regions of sequence diversity of the homologous nucleic acids.

9. The method of claim 1, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to one or more specific regions of sequence diversity of the homologous nucleic acids.

10. The method of claim 1, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property, wherein the selected one or more recombined nucleic acids encode industrial enzymes.

11. The method of claim 1, wherein members of the set of family gene shuffling oligonucleotides overlap.

12. The method of claim 1, wherein elongating the set of family gene shuffling oligonucleotides is performed with a polymerase or a ligase.

13. The method of claim 1, wherein the set of family gene shuffling oligonucleotides encodes an evolutionary intermediate nucleic acid.

14. 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.

15. The method of claim 14, wherein the reannealed recombined nucleic acids are recombined.

16. The method of claim 14, further comprising recombining the resulting selected recombined nucleic acids.

17. The method of claim 16, further comprising selecting the resulting multiple recombined nucleic acids for the desired trait or property.

18. 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.

19. The method of claim 18, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property prior to repeating (i) and (ii).

20. The method of claim 18, wherein the population of recombined nucleic acids has greater molecular diversity than the homologous nucleic acids.

21. The method of claim 18, wherein at least one nucleic acid in the population of recombined nucleic acids includes regions of diversity from multiple homologous nucleic acids.

22. The method of claim 18, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids based on diversity of the population of recombined nucleic acids.

23. The method of claim 18, wherein the chemically synthesized set of family gene shuffling oligonucleotides comprises a set of crossover family diversity oligonucleotides.

24. The method of claim 18, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to multiple regions of sequence diversity of the homologous nucleic acids.

25. The method of claim 18, wherein at least some oligonucleotides in the chemically synthesized set of family gene shuffling oligonucleotides correspond to one or more specific regions of sequence diversity of the homologous nucleic acids.

26. The method of claim 18, further comprising selecting one or more recombined nucleic acids from the population of recombined nucleic acids for a desired enzymatic property, wherein the selected one or more recombined nucleic acids encode industrial enzymes.

27. The method of claim 18, wherein members of the set of family gene shuffling oligonucleotides overlap.

28. The method of claim 18, wherein elongating the set of family gene shuffling oligonucleotides is performed with a polymerase or a ligase.

29. The method of claim 18, wherein the set of family gene shuffling oligonucleotides encodes an evolutionary intermediate nucleic acid.

30. 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.

31. The method of claim 30, wherein the reannealed recombined nucleic acids are recombined.

32. The method of claim 30, further comprising recombining the resulting selected recombined nucleic acids.

33. The method of claim 32, further comprising selecting the resulting multiple recombined nucleic acids for the desired trait or property.