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

• US 6,444,468 B1
• Filed: 11/28/2000
• Issued: 09/03/2002
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C., Crameri, Andreas
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/724,958
• Time in Patent Office: 644 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: 506/1
• 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 15/1034 Isolating an individual clone by screening libraries
• 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 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
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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 for obtaining a chimeric polynucleotide comprising:

a) treating a sample comprising different double-stranded template polynucleotides wherein said different template polynucleotides contain areas of identity and areas of heterology under conditions which provide for the cleavage of said double stranded template polynucleotides into random double-stranded template fragments of a desired size;

b) denaturing the resultant random double-stranded template fragments contained in the treated sample produced by step (a) into single-stranded fragments;

c) incubating the resultant single-stranded fragments with polymerase under conditions which provide for the annealing of the target single-stranded fragments at the areas of identity and the formation of chimeric double-stranded polynucleotide sequences comprising template polynucleotide sequences; and

d) repeating steps (b) and (c) at least twice;

e) selecting or screening the chimeric double stranded polynucleotide sequences to identify a chimeric double stranded polynucleotide sequence having a desired functional property.

2. The method of claim 1 wherein the concentration of a specific double-stranded template fragment in the mixture of double-stranded fragments in step (a) is less than 1% by weight of the total DNA.

3. The method of claim 1 where the number of different specific double-stranded template fragments in step (a) comprises at least about 100.

4. The method of claim 1 wherein the size of the double-stranded template fragments is from about 5 bp to 5 kb.

5. The method of claim 1 wherein the size of the chimeric double-stranded polynucleotide comprises from 50 bp to 100 kb.

6. The method of claim 1, wherein step (b) and (c) are repeat 2-50 times.

7. The method of claim 1, wherein step (a) comprises treating the sample with DNase.

8. The method of claim 1, wherein the denaturing is performed at 80-100°

C.

9. The method of claim 1, wherein the denaturing is performed at 90-96°

C.

10. The method of claim 1, wherein the annealing is performed at 40-65°

C.

11. The method of claim 1, wherein the annealing is performed in the presence of PEG.

12. The method of claim 1, wherein the annealing is performed at a salt concentration of 10-100 nM.

13. The method of claim 1, wherein the polymerase is taq.

14. The method of claim 1, wherein the polymerase is Klenow.

15. The method of claim 1, wherein steps (b) and (c) are repeated until a chimeric double stranded polynucleotide of 500-50 kb is produced.

16. The method of claim 1, wherein steps (b) and (c) are repeated until a concatemeric chimeric double stranded polynucleotide is produced.

17. The method of claim 16, further comprising digesting the concatemeric chimeric double stranded polynucleotide to produce isolated copies of the repeat unit forming the concatmer.

18. The method of claim 1, wherein the method is repeated to produced a plurality of chimeric double stranded polynucleotides, and the chimeric double stranded polynucleotides are cloned into a vector, transformed into host cells and expressed.

19. The method of claim 1, further comprising selecting the chimeric double stranded polynucleotides or their expression product for a desired functional property.

20. The method of claim 19, performed iteratively with a supopulation of chimeric double stranded polynucleotide having a desired functional property or encoding an expression product with a desired functional property from one iteration providing the different double stranded template polynucleotides for the next iteration.

21. The method of claim 20, wherein the different double stranded template polynucleotides in the next iteration further comprise fragments of wildtype templates to remove silent mutations from the chimeric double sanded polynucleotides having the desired functional property.

22. The method of claim 1, wherein the desired functional property of the expression product ability to bind a ligand.

23. The method of claim 1, wherein the desired functional property of the expression product is drug resistance.

24. The method of claim 1, wherein the desired functional property is a therapeutic activity.

25. The method of claim 1, wherein the desired functional property is suitability as an agent for gene therapy.

26. The method of claim 1, wherein the desired functional property is suitability as an agent for anti-neoplastic therapy.

27. The method of claim 1, wherein the desired functional property is suitability as an agent for DNA-based vaccination.

28. The method of claim 1, wherein the desired fictional property is an enhanced biological activity relative to an existing polynucleotide variant.

29. The method of claim 1, wherein the selecting or screening step isolates a chimeric double stranded polynucleotide encoding an enzyme.

30. The method of claim 1, wherein the selecting or screening step isolates a chimeric double stranded polynucleotide encoding an antibody chain.

31. The method of claim 1, wherein the selecting or screening step isolates a chimeric double-stranded polynucleotide encoding a viral coat protein.

32. The method of claim 1, wherein the regions of heterology are point mutations.

33. The method of claim 1, wherein the different double stranded template polynucleotides are at least 50 bp.

34. The method of claim 1, wherein the different double stranded template polynucleotides are obtained from a natural source.

35. The method of claim 34, wherein the different double stranded template polynucleotides comprises allelic variants.

36. The method of claim 34, wherein the different double stranded template polynucleotides comprise species variants.

37. The method of claim 1, wherein the different double stranded template polynucleotides are obtained by mutagenesis.

38. The method of claim 1, wherein the different template polynucleotide sequences are of unknown sequence.

39. The method of claim 1, further comprising adding synthetic oligonucleotides, PCR fragments or whole genes to the sample.

40. The method of claim 1, wherein the sample is free of primers other than generated by the treating step.

41. The method of claim 1, wherein the different template polynucleotides comprise at least eight different templates.

42. The method of claim 1, further comprising performing error-prone PCR on at least the different template polynucleotides.

43. The method of claim 1, wherein the different template polynucleotides comprise a human polynucleotide.

44. The method of claim 1, wherein the different template polynucleotides comprise a mouse polynucleotide.

45. The method of claim 1, wherein the different template polynucleotides comprise a human polynucleotide and a mouse polynucleotide.

46. The method of claim 1, wherein the different template polynucleotides comprise bacterial polynucleotides.

47. The method of claim 1, wherein the different template polynucleotides comprise plant polynucleotides.

48. The method of claim 1, wherein the different template polynucleotides comprise yeast polynucleotides.

49. The method of claim 1, wherein the different template polynucleotides comprise comprise animal polynucleotides.

50. The method of claim 1, wherein the different template polynucleotides comprise viral polynucleotides.

51. The method of claim 1, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.

52. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting, the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the cycles of denaturation are performed at 80-100°

C.

53. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the annealing is performed at 40-65°

C.

54. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the annealing is performed in the presence of PEG.

55. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the annealing is performed at a salt concentration of 10-100 nM.

56. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the extension is performed using taq polymerase.

57. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the extension is performed using Klenow.

58. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the shuffled polynucleotides have a length of 500-50 kb.

59. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein the shuffled polynucleotides comprises concatemers.

60. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein sequences from at least eight polynucleotide variants are present.

61. A method of shuffling polynucleotides, comprising:

conducting multiple cycles of denaturation, annealing and extenion on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants 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 shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property;

wherein sequence are from polynucleotide variants of unknown sequence.

62. A method of shuffling polynucleotides, comprising:

randomly cleaving a mixed population of polynucleotide vanants to produce fragments;

conducting multiple cycles of denaturation, annealing and extension on the fragments under conditions whereby one strand of a fragment serves as a template for extension of a strand from another fragment to generate a population of shuffled polynucleotides; and

screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property.