Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

• US 6,518,065 B1
• Filed: 10/25/2000
• Issued: 02/11/2003
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/696,313
• Time in Patent Office: 839 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/5
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

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.

Claims

1. A method of identifying a recombinant nucleic acid with a desired property, the method comprising:

(a) providing a plurality of random fragments of at least a first and a second nucleic acid;

(b) recombining the random fragments one or more times to produce at least one recombinant nucleic acid; and

,

(c) identifying at least one recombinant nucleic acid with the desired property.

2. The method of claim 1, wherein the random fragments comprise DNA fragments.

3. The method of claim 1, comprising providing the random fragments by digesting the at least first and second nucleic acids with a nuclease.

4. The method of claim 3, wherein the nuclease comprises DNAse I.

5. The method of claim 1, comprising providing the random fragments by one or more of physical shearing, chemical cleavage or a polymerase chain reaction.

6. The method of claim 5, wherein the polymerase chain reaction comprises a partial PCR extension or PCR stuttering.

7. The method of claim 1, wherein the random fragments of the at least first and second nucleic acids are recombined in vitro.

8. The method of claim 1, wherein the random fragments of the at least first and second nucleic acids are recombined in vivo.

9. The method of claim 1, wherein the at least first and second nucleic acids comprise related nucleic acids.

10. The method of claim 1, wherein the at least first and second nucleic acids comprise homologous nucleic acids.

11. The method of claim 1, wherein the at least first and second nucleic acids comprise members of a family of nucleic acids.

12. The method of claim 1, wherein the at least first and second nucleic acids comprise allelic variants.

13. The method of claim 1, wherein the at least first and second nucleic acids comprise species variants.

14. The method of claim 1, wherein one or more of the at least first and second nucleic acids comprise wild type nucleic acid.

15. The method of claim 1, comprising recombining the random fragments of the at least first and second nucleic acids by annealing a population of random fragments of the first and second nucleic acids, wherein at least one fragment serves as a template for extension of another fragment, and extending the annealed fragments with a polymerase.

16. The method of claim 15, comprising recombining the random fragments of the at least first and second nucleic acids in an amplification reaction.

17. The method of claim 16, comprising extending the annealed fragments with a thermostable polymerase.

18. The method of claim 1, wherein providing the random fragments of the at least first and second nucleic acids comprises:

providing a first population of overlapping nucleic acid fragments corresponding to a plurality of subsequences of the first nucleic acid and a second population of overlapping nucleic acid fragments corresponding to a plurality of subsequences of the second nucleic acid.

19. The method of claim 18, wherein the first and second nucleic acid encode a first and a second polypeptide.

20. The method of claim 1, further comprising recombining the at least one recombinant nucleic acid with the first or the second nucleic acid, thereby backcrossing the recombinant nucleic acid to the first or the second nucleic acid.

21. The method of claim 20, comprising recombining the at least one recombinant nucleic acid with an excess of the first or the second nucleic acid.

22. The method of claim 20, further comprising identifying the at least one recombinant nucleic acid with the desired property prior to recombining the at least one recombinant nucleic acid with the first or the second nucleic acid.

23. The method of claim 20, further comprising identifying at least one backcrossed recombinant nucleic acid with a desired property.

24. A method of making a recombinant nucleic acid, the method comprising:

(a) recombining at least a first and a second nucleic acid one or more times in vitro to produce at least one recombinant nucleic acid; and

,

(b) recombining the at least one recombinant nucleic acid with the first or the second nucleic acid, thereby backcrossing the recombinant nucleic acid to the first or the second nucleic acid.

25. The method of claim 24, comprising recombining the at least one recombinant nucleic acid with an excess of the first or the second nucleic acid.

26. The method of claim 24, further comprising identifying at least one recombinant nucleic acid with a desired property prior to recombining the at least one recombinant nucleic acid with the first or the second nucleic acid.

27. The method of claim 24, further comprising identifying at least one backcrossed recombinant nucleic acid with a desired property.

28. The method of claim 24, wherein the at least first and second nucleic acids comprise DNA sequences.

29. The method of claim 24, wherein the first or second nucleic acid comprises a wild type nucleic acid.

30. The method of claim 24, wherein the at least first and second nucleic acids comprise related nucleic acids.

31. The method of claim 24, wherein the at least first and second nucleic acids comprise homologous nucleic acids.

32. The method of claim 24, wherein the at least first and second nucleic acids comprise allelic variants.

33. The method of claim 24, wherein the at least first and second nucleic acids comprise species variants.

34. The method of claim 24, wherein the at least first and second nucleic acids comprise members of a family of nucleic acids.

35. The method of claim 24, comprising recombining the first and second nucleic acids by annealing a population of nucleic acid fragments, wherein at least one fragment serves as a template for extension of another fragment, and extending the annealed fragments with a polymerase.

36. The method of claim 35, comprising recombining the first and second nucleic acids in an amplification reaction.

37. The method of claim 36, comprising extending the annealed fragments with a thermostable polymerase.

38. The method of claim 24, wherein recombining the at least first and second nucleic acids comprises:

(a) providing a first population of overlapping nucleic acid fragments corresponding to a plurality of subsequences of the first nucleic acid and a second population of overlapping nucleic acid fragments corresponding to a plurality of subsequences of the second nucleic acid; and

(b) recombining a plurality of overlapping nucleic acid fragments from the first population with a plurality of overlapping nucleic acid fragments from the second population, thereby producing a library of chimeric polynucleotides comprising recombinant nucleic acids.

39. The method of claim 38, wherein the first and second nucleic acids encode a first and a second polypeptide.

40. The method of claim 39, further comprising expressing the library of chimeric polynucleotides to produce a population of chimeric polypeptides.

41. The method of claim 38, wherein the first or second populations of nucleic acid fragments, or both the first and second population of nucleic acid fragments are produced by enzymatic digestion of the first or second nucleic acids, or by enzymatic digestion of both the first and second nucleic acids.

42. The method of claim 38, wherein the first or second populations of nucleic acid fragments, or both the first and second population of nucleic acid fragments are produced by sonication of the first or second nucleic acid, or by sonication of both the first and second nucleic acids.

43. The method of claim 38, wherein the first or second populations of nucleic acid fragments, or both the first and second population of nucleic acid fragments are produced by mechanical shearing of the first or second nucleic acid, or by mechanical shearing of both the first and second nucleic acids.

44. The method of claim 38, wherein the first or second populations of nucleic acid fragments, or both the first and second population of nucleic acid fragments are produced by partial amplification of the first or second nucleic acid, or by partial amplification of both the first and second nucleic acids.

45. The method of claim 38, wherein the first or second populations of nucleic acid fragments, or both the first and second population of nucleic acid fragments are produced by synthesis of oligonucleotides corresponding to subsequences of the first or second nucleic acid, or by synthesis of oligonucleotides corresponding to subsequences of both the first and second nucleic acids.