Oligonucleotide mediated nucleic acid recombination

• US 6,479,652 B1
• Filed: 07/27/2000
• Issued: 11/12/2002
• Est. Priority Date: 01/19/1999
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Ness, Jon E., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C., Bass, Steven H., Crameri, Andreas, Welch, Mark
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/626,595
• Time in Patent Office: 838 Days
• Field of Search: 536/23.1, 536/24.3, 435/6, 435/91.2, 435/440, 935/76, 935/77, 935/7.D
• US Class Current: 536/23.1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 2740/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)

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 composition, comprising:

a set of nucleic acids, comprising;

a first subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a first target nucleic acid; and

,

a second subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a second target nucleic acid;

wherein the first and second target nucleic acids encode non-identical proteins and comprise a plurality of regions of difference, and

wherein the first and second subsets of chemically synthesized oligonucleotide members correspond to the regions of difference, and the first and second subsets are present in substantially non-equimolar amounts.

2. The composition of claim 1, wherein the members of the first subset which correspond to the regions of difference are present in the composition at a ratio of about 60 percent to about 40 percent members of the second set which correspond to the regions of difference, or higher.

3. The composition of claim 1, wherein the plurality of regions of difference comprise a plurality of non-overlapping subsequence regions of the first or second target nucleic acid.

4. The composition of claim 1, wherein the members of the first or second subsets each have a sequence or subsequence identical to at least one subsequence from at least one of the selected target sequences.

5. The composition of claim 1, wherein the members of the first or second subsets comprise a plurality of homologous oligonucleotides corresponding to a plurality of regions of homology between the first and second target sequences, wherein each of the plurality of homologous oligonucleotides comprise at least one variant subsequence.

6. The composition of claim 1, further comprising one or more of:

a polymerase, a thermostable DNA polymerase, a nucleic acid synthesis reagent, a buffer, a salt, magnesium, Taq polymerase, Klenow polymerase, dATP, dTTP, dGTP, dCTP, or one or more nucleic acid comprising one or more of the plurality of members of the selected set of homologous target sequences.

7. The composition of claim 1, wherein the set of nucleic acids is provided to the composition by aligning a plurality of homologous target sequences, determining at least one region of identity and at least one region of variance and synthesizing the set of oligonucleotides to encode at least a portion of the at least one region of identity, or at least a portion of the at least one region of variance, or at least a portion of both the at least one region of identity and the at least one region of variance.

8. The composition of claim 1, wherein the set of nucleic acids comprises a plurality of members collectively comprising at least 5 sequence diversity domains.

9. The composition of claim 1, wherein the set of nucleic acids comprises 10 or more sequence diversity domains.

10. The composition of claim 1, wherein the set of nucleic acids comprises a set of crossover family diversity oligonucleotides, each comprising a plurality of sequence a diversity domains, with at least a first of the sequence diversity domains corresponding to the first target nucleic acid and a second of the diversity domains corresponding to the second target nucleic acid.

11. The composition of claim 9, wherein the sequence diversity domains correspond to adjacent sequence regions on the first and second target nucleic acids when sequences corresponding to the target nucleic acids are aligned.

12. The composition of claim 1, wherein the oligonucleotide members of the first subset which correspond to the regions of difference are present in the composition at a ratio of about 70 percent to about 30 percent members of the second set which correspond to the regions of difference, or higher.

13. The composition of claim 1, wherein the oligonucleotide members of the first subset which correspond to the regions of difference are present in the composition at a ratio of about 80 percent to about 20 percent members of the second set which correspond to the regions of difference, or higher.

14. The composition of claim 1, wherein the oligonucleotide members of the first subset which correspond to the regions of difference are present in the composition at a ratio of about 90 percent to about 10 percent members of the second set which correspond to the regions of difference, or higher.

15. The composition of claim 1, wherein the first and second sets of oligonucleotides together comprise a plurality of members collectively comprising at least 10 sequence diversity domains.

16. The composition of claim 1, wherein the first and second sets of oligonucleotides together comprise a plurality of members collectively comprising at least 20 sequence diversity domains.

17. The composition of claim 1, wherein the first and second sets of oligonucleotides together comprise a plurality of members collectively comprising at least 50 sequence diversity domains.

18. The composition of claim 1, wherein the first and second sets of oligonucleotides together comprise a plurality of members collectively comprising at least 75 sequence diversity domains.

19. The composition of claim 1, wherein the first and second sets of oligonucleotides together comprise a plurality of members collectively comprising at least 100 sequence diversity domains.

20. The composition of claim 1, wherein the first or second subset is present at a molar ratio of 50% or greater of the set of nucleic acids.

21. The composition of claim 1, wherein the first or second subset is present at a molar ratio of 60% or greater of the set of nucleic acids.

22. The composition of claim 1, wherein the first or second subset is present at a molar ratio of 70% or greater of the set of nucleic acids.

23. The composition of claim 1, wherein the first or second subset is present at a molar ratio of 80% or greater of the set of nucleic acids.

24. The composition of claim 1, wherein the first or second subset is present at a molar ratio of 90% or greater of the set of nucleic acids.

31. The composition of claim 1, wherein the first and second target nucleic acids are 50% or more identical in sequence when aligned for maximum correspondence.

32. The composition of claim 1, wherein the first and second target nucleic acids are 80% or more identical in sequence when aligned for maximum correspondence.

33. The composition of claim 1, wherein the first and second target nucleic acids are 90% or more identical in sequence when aligned for maximum correspondence.

34. The composition of claim 1, wherein the first and second target nucleic acids are 95% or more identical in sequence when aligned for maximum correspondence.

35. The composition of claim 1, wherein the members of the first and second subset are substantially similar in length.

36. The composition of claim 35, wherein the members of the first and second subset are about 20 or more nucleotides in length.

37. The composition of claim 35, wherein the members of the first and second subset are about 30 or more nucleotides in length.

38. The composition of claim 35, wherein the members of the first and second subset are about 40 or more nucleotides in length.

39. The composition of claim 35, wherein the members of the first and second subset are about 50 or more nucleotides in length.

40. The composition of claim 35, wherein the members of the first and second subset are about 60 or more nucleotides in length.

41. The composition of claim 35, wherein the members of the first and second subset are about 70 or more nucleotides in length.

42. The composition of claim 35, wherein the members of the first and second subset are about 80 or more nucleotides in length.

43. The composition of claim 35, wherein the members of the first and second subset are about 90 or more nucleotides in length.

44. The composition of claim 35, wherein the members of the first and second subset are about 100 or more nucleotides in length.

45. The composition of claim 1, wherein the members of the first or second subsets comprise overlapping oligonucleotide members.

46. The composition of claim 45, wherein the members of the first or second subsets overlap by about 5 or more bases.

47. The composition of claim 45, wherein the members of the first or second subsets overlap by about 10 or more bases.

48. The composition of claim 45, wherein the members of the first or second subsets overlap by about 20 or more bases.

55. The composition of claim 1, wherein the set of nucleic acids comprises one or more chimeraplasts.

56. The composition of claim 1, wherein the set of nucleic acids comprises one or more DNA oligonucleotides.

57. The composition of claim 1, wherein the set of nucleic acids comprises one or more oligonucleotide that comprises one or more non-standard nucleotide.

58. The composition of claim 1, wherein the set of nucleic acids comprises one or more oligonucleotide that comprises one or more methylated nucleotide.

61. The composition of claim 1, wherein the set of nucleic acids comprises one or more cross-over oligonucleotide.

62. The composition of claim 61, wherein the first and second target nucleic acids display low sequence similarity.

63. The composition of claim 61, wherein the first and second target nucleic acids display low sequence similarity and wherein the cross-over oligonucleotide comprises a first subsequence corresponding to the first target nucleic acid and a second subsequence corresponding to the second target nucleic acid.

64. The composition of claim 1, wherein the first or second target nucleic acid are gene blended nucleic acids.

65. The composition of claim 1, wherein the first or second target nucleic acid comprise a frog gene or a human gene.

66. The composition of claim 1, wherein the first or second target nucleic acid encodes one or more of:

erythropoietin, insulin, a peptide hormone, human growth hormone, a growth factor, a cytokine, epithelial Neutrophil Activating Peptide-78, GROα

/MGSA, GROβ

, GROγ

, MIP-1α

, MIP-16, MCP-1, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, an interferon, an interleukin, a keratinocyte growth factor, a leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGEF, G-CSF, a transcriptional activator, an expression activator, an inflammatory molecule, a growth factor receptor, an oncogene product, IL-1, IL-2, IL-8, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-cs, TGF-P, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44;

Mos, Ras, Raf, Met, p53, Tat, Fos, Myc, Jun, Myb, Rel, a steroid hormone receptor, an Rnase, Onconase, EDN, a vaccine protein, a vaccine protein derived from an infectious fungus, a vaccine protein derived from a bacteria, a vaccine protein derived from a virus, an enzyme, a monooxygenase, a p450, a protease, a nuclease, a lipase, Alpha-I antitrypsin, Angiostatin, Antihemolytic factor, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, an Atrial peptide, a C-X-C chemokines, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-I0, GCP-2, NAP-4, SDF- 1, PF4, MIG), Calcitonin, a CC chemokine, Monocyte chemoattractant protein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractant protein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, Collagen, Colony stimulating fact (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, Factor LX, Factor VII, Factor VIII, Factor X, Fibrinogen, Fibronectin, Glucocerebrosidase, Gonadotropin, a Hedgehog protein, Hemoglobin, Hirudin, Human serum albumin, Lactoferrin, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), Osteogenic protein, Parathyroid hormone, Protein A, Protein G, Relaxin, Renin, Salmon calcitonin, Salmon growth hormone, Soluble complement receptor I, Soluble I-CAM 1, a soluble interleukin receptor, a soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, a Superantigen, a Staphylococcal enterotoxins, SEA, SEB, SEC1SEC2, SEC3, SED, SEE, Toxic shock syndrome toxin, exfoliating toxin A, exfoliating toxin B, Pyrogenic exotoxin A, Pyrogenic exotoxin B, Pyrogenic exotoxin C, M. arthritides mitogen, Superoxide dismutase, Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNF alpha), Urokinase, a defensin, EF40, a peptide antibiotic, or a peptide insecticidal protein.

67. The composition of claim 1, wherein the first subset members comprise chemically synthesized oligonucleotides and the second subset members comprise chemically synthesized oligonucleotides.

68. The composition of claim 1, wherein the first subset members comprise a plurality of oligonucleotides of a selected length and the second subset members comprise a plurality of oligonucleotides of a selected length.

71. The method of claim 1, wherein the first subset collectively corresponds to 50% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 50% or more of the second target nucleic acid, or both.

72. The method of claim 1, wherein the first subset collectively corresponds to 60% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 60% or more of the second target nucleic acid, or both.

73. The method of claim 1, wherein the first subset collectively corresponds to 70% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 70% or more of the second target nucleic acird, or both.

74. The method of claim 1, wherein the first subset collectively corresponds to 80% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 80% or more of the second target nucleic acid, or both.

75. The method of claim 1, wherein the first subset collectively corresponds to 90% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 90% or more of the second target nucleic acid, or both.

76. The method of claim 1, wherein the first subset collectively corresponds to 100% of the first target nucleic acid, or wherein the second subset collectively corresponds to 100% of the second target nucleic acid, or both.

25. A composition, comprising:

a set of nucleic acids, comprising;

a first subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a first target nucleic acid;

a second subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a second target nucleic acid;

wherein the first and second target nucleic acids comprise a plurality of regions of difference, the first and second subsets of oligonucleotide members correspond to the regions of difference, and the first and second subsets are present in substantially non-equimolar amounts; and

a third subset of chemically sythesized oligonucleotide members which collectively correspond to all or a substantial portion of a third target member, which third subset is present in the composition in an amount that is substantially non-equimolar as compared to the first or second subsets.

26. The composition of claim 25, wherein the first, second or third subset is present at a molar ratio of 50% or greater of the overall set of nucleic acids.

27. The composition of claim 25, wherein the first, second or third subset is present at a molar ratio of 60% or greater of the overall set of nucleic acids.

28. The composition of claim 25, wherein the first, second or third subset is present at a molar ratio of 70% or greater of the overall set of nucleic acids.

29. The composition of claim 25, wherein the first, second or third subset is present at a molar ratio of 80% or greater of the overall set of nucleic acids.

30. The composition of claim 25, wherein the first, second or third subset is present at a molar ratio of 90% or greater of the overall set of nucleic acids.

69. The composition of claim 25, wherein the first subset members comprise chemically synthesized oligonucleotides and the second subset members comprise chemically synthesized oligonucleotides.

70. The composition of claim 25, wherein the first subset members comprise a plurality of oligonucleotides of a selected length and the second subset members comprise a plurality of oligonucleotides of a selected length.

77. The method of claim 25, wherein the first subset collectively corresponds to 50% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 50% or more of the second target nucleic acid, or both.

78. The method of claim 25, wherein the first subset collectively corresponds to 60% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 60% or more of the second target nucleic acid, or both.

79. The method of claim 25, wherein the first subset collectively corresponds to 70% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 70% or more of the second target nucleic acid, or both.

80. The method of claim 25, wherein the first subset collectively corresponds to 80% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 80% or more of the second target nucleic acid, or both.

81. The method of claim 25, wherein the first subset collectively corresponds to 90% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 90% or more of the second target nucleic acid, or both.

82. The method of claim 25, wherein the first subset collectively corresponds to 100% of the first target nucleic acid, or wherein the second subset collectively corresponds to 100% of the second target nucleic acid, or both.

49. A composition, comprising:

a set of nucleic acids, comprising;

a first subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a first target nucleic acid;

a second subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a second target nucleic acid;

wherein the first and second target nucleic acids comprise a plurality of regions of difference, the first and second subsets of oligonucleotide or nucleic acid fragment members correspond to the regions of difference, and the first and second subsets are present in substantially non-equimolar amounts; and

,

wherein the first subset comprises a first oligonucleotide corresponding to at least one of the regions of difference, and wherein the second subset comprises a second oligonucleotide corresponding to the at least one region of difference, wherein the first and second oligonucleotides comprise subsequences of identity flanking a subsequence of difference.

50. The composition of claim 49, wherein at least one of the subsequences of identity is 5 or more nucleotides in length.

51. The composition of claim 49, wherein at least one of the subsequences of identity is 10 or more residues in length.

52. The composition of claim 49, wherein at least one of the subsequences of identity is 15 or more residues in length.

53. The composition of claim 49, wherein at least one of the subsequences of identity is 20 or more residues in length.

54. The composition of claim 49, wherein at least one of the subsequences of identity is 50 or more residues in length.

83. The composition of claim 49, wherein the first subset collectively corresponds to 50% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 50% or more of the second target nucleic acid, or both.

84. The composition of claim 49, wherein the first subset collectively corresponds to 60% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 60% or more of the second target nucleic acid, or both.

85. The composition of claim 49, wherein the first subset collectively corresponds to 70% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 70% or more of the second target nucleic acid, or both.

86. The composition of claim 49, wherein the first subset collectively corresponds to 80% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 80% or more of the second target nucleic acid, or both.

87. The composition of claim 49, wherein the first subset collectively corresponds to 90% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 90% or more of the second target nucleic acid, or both.

88. The composition of claim 49, wherein the first subset collectively corresponds to 100% of the first target nucleic acid, or wherein the second subset collectively corresponds to 100% of the second target nucleic acid, or both.

59. A composition, comprising:

a set of nucleic acids, comprising;

a first subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a first target nucleic acid;

a second subset of chemically synthesized oligonucleotide members which collectively correspond to at least a substantial portion of a second target nucleic acid;

wherein the first and second target nucleic acids comprise a plurality of regions of difference, the first and second subsets of oligonucleotide members correspond to the regions of difference, and the first and second subsets are present in substantially non-equimolar amounts; and

,

wherein at least one variant member of the set of nucleic acids comprises a sequence which corresponds to the first target nucleic acid, the second target nucleic acid, or both the first and second target nucleic acid, which sequence comprises one or more sequence variation as compared to either the first or second target nucleic acid.

60. The composition of claim 59, wherein the variant member is selected from the group consisting of:

a cross-over oligonucleotide, a codon-varied oligonucleotide, an oligonucleotide adjusted for codon bias, and a mutant oligonucleotide.

89. The composition of claim 59, wherein the first subset collectively corresponds to 50% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 50% or more of the second target nucleic acid, or both.

90. The composition of claim 59, wherein the first subset collectively corresponds to 60% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 60% or more of the second target nucleic acid, or both.

91. The composition of claim 59, wherein the first subset collectively corresponds to 70% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 70% or more of the second target nucleic acid, or both.

92. The composition of claim 59, wherein the first subset collectively corresponds to 80% or more of the first target nucleic acid, or wherein the second subset collectively correpsonds to 80% or more of the second target nucleic acid, or both.

93. The composition of claim 59, wherein the first subset collectively corresponds to 90% or more of the first target nucleic acid, or wherein the second subset collectively corresponds to 90% or more of the second target nucleic acid, or both.

94. The composition of claim 60, wherein the first subset collectively corresponds to 100% of the first target nucleic acid, or wherein the second subset collectively corresponds to 100% of the second target nucleic acid, or both.