Methods for generating recombined polynucleotides

• US 6,159,687 A
• Filed: 03/18/1998
• Issued: 12/12/2000
• Est. Priority Date: 03/18/1997
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Nordisk A/S
• Inventors: Vind, Jesper
• Primary Examiner(s): YUCEL, IREM
• Application Number:
  US09/040,697
• Time in Patent Office: 1,000 Days
• Field of Search: 435/4, 435/6, 435/69.1, 435/69.4-69.7, 435/91.1, 435/91.2, 435/91.5, 435/440, 435/455, 435/471, 435/476, 536/24.3, 536/24.31, 536/24.32
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups

Abstract

A method for in vitro construction of a library of recombined homologous polynucleotides from a number of different starting DNA templates and primers by induced template shifts during an polynucleotide synthesis is described, whereby

A. extended primers are synthesized by

a) denaturing the DNA templates

b) annealing primers to the templates,

c) extending the said primers by use of a polymerase,

d) stop the synthesis, and

e) separate the extended primers from the templates,

B. a template shift is induced by

a) isolating the extended primers from the templates and repeating steps A.b) to A.e) using the extended primers as both primers and templates, or

b) repeating steps A.b) to A.e),

C. this process is terminated after an appropriate number of cycles of process steps A. and B.a), A. and B.b), or combinations thereof.

Optionally the polynucleotides are amplified in a standard PCR reaction with specific primers to selectively amplify homologous polynucleotides of interest.

Claims

1. A method of shuffling polynucleotides, comprising

(a) providing an isolated sense strand from at least a first variant form of a polynucleotide, and an isolated antisense strand from at least a second variant form of the polynucleotide, wherein the isolated sense and antisense strands are starting substrates to be shuffled;

and

(b) shuffling the isolated sense and antisense strands provided in step (a) by contacting the strands with at least one primer or one pair of primers and conducting a multi-cyclic polynucleotide extension reaction wherein

(i) in at least one cycle, the primer(s) anneal to the isolated strands and generate extended primers; and

(ii) in at least one subsequent cycle, the extended primers generated in a previous cycle act as both primers and templates to form further extended primers,

whereby the polynucleotide extension is continued for sufficient cycles until the further extended primers includes recombinant forms of the shuffled polynucleotides.

2. The method of claim 1, wherein the isolated strands are labeled with biotin.

3. The method of claim 1, wherein each sense and antisense strand comprises a pool of diverse forms of a polynucleotide to be shuffled.

4. The method of claim 1, wherein the first and second forms of the polynucleotide variants exhibit more than 50% sequence identity.

5. The method of claim 4, wherein the polynucleotide variants exhibit more than 70% sequence identity.

6. The method of claim 5, wherein the polynucleotide variants exhibit more than 90% sequence identity.

7. The method of claim 6, wherein the polynucleotide variants exhibit more than 95% sequence identity.

8. The method of claim 1, wherein the at least one primer is completely random.

9. The method of claim 1, wherein the at least one primer or at least a pair of primers is (are) partly random.

10. A method of identifying polypeptides exhibiting a desired property, comprising the method of claim 1, further comprising step (c) expressing and screening a polypeptide encoded by the shuffled polynucleotide for a desired property.

11. The method of claim 10, wherein the property is an enzymatic activity.

12. The method of claim 1, wherein the at least first and second variants forms are from naturally occurring organisms of different species.

13. The method of claim 1, further comprising separating the extended primers from the starting substrates.

14. The method of claim 1, wherein at least one extension cycle is conducted under conditions of incomplete elongation.

15. The method of claim 1, further comprising selecting the starting substrates to be shuffled.

16. A method for shuffling polynucleotides, comprising

(a) providing at least two variant forms of a polynucleotide as starting substrates to be shuffled;

and

(b) shuffling the at least two variant polynucleotides by contacting the variants with a population of completely or partially random primers; and

conducting a multi-cyclic polynucleotide extension reaction wherein

(i) in at least one cycle, the primers anneal to the variants to generate extended primers, wherein the at least one cycle is conducted with a shorter extension time than required for complete extension of the starting substrates; and

(ii) in at least one subsequent cycle, the extended primers generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations forming annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form further extended primers;

whereby the polynucleotide extension is continued for sufficient cycles until the further extended primers includes recombinant forms of the shuffled polynucleotide.

17. The method of claim 16, wherein the starting substrates are labeled, and separated from the extended primers generated in step (b) by use of the label.

18. The method of claim 16, wherein the multi-cyclic polynucleotide extension reaction is conducted under conditions promoting misincorporation of nucleotides.

19. The method of claim 16, wherein the starting substrates have a length of 50 bp to 20 kb.

20. The method of claim 16, wherein the multi-cyclic polynucleotide extension reaction is conducted with a thermostable DNA polymerase.

21. The method of claim 20, wherein the polymerase is Taq, Amplitaq, Vent, or Pwo.

22. The method of claim 16, wherein the multi-cyclic polynucleotide extension reaction is conducted with a polymerase selected from the group consisting of T4 polymerase, T7 polymerase, E. coli DNA polymerase I and the Klenow fragment of DNA polymerase I, and wherein said polymerase is added to the reaction mixture after each cycle.

23. The method of claim 16, wherein the multi-cyclic polynucleotide extension reaction is conducted in the presence of an RNA polymerase.

24. The method of claim 16, wherein said polymerase is an error-prone polymerase.

25. The method of claim 16, wherein the starting substrates are natural variants.

26. The method of claim 16, wherein at least two starting substrates exhibit more than 50% sequence identity.

27. The method of claim 26, wherein the polynucleotide variants exhibit more than 70% sequence identity.

28. The method of claim 27, wherein the polynucleotide variants exhibit more than 90% sequence identity.

29. The method of claim 27, wherein the polynucleotide variants exhibit more than 95% sequence identity.

30. The method of claim 16, wherein the primers are labeled.

31. The method of claim 30, wherein the label is biotin or digoxigenin.

32. The method of claim 16, wherein the primers are 6 to 200 bp in length.

33. The method of claim 32, wherein the primers are 10 to 70 bp.

34. The method of claim 33, wherein the primers are 15 to 40 bp.

35. The method of claim 16, wherein the substrates are from naturally occurring organisms of different species.

36. The method of claim 16, wherein the starting substrates are linear DNA fragments generated by a PCR reaction.

37. The method of claim 16, wherein the starting substrates are cloned into vectors.

38. The method of claim 16, wherein the starting substrates encode variant forms of an enzyme.

39. The method of claim 38, wherein enzyme is selected from the group consisting of carbonyl hydrolase, carbohydrase, an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.

40. The method of claim 16, wherein the starting substrates encode variant forms of a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

41. The method of claim 16, wherein the starting substrates have a function selected from the group consisting of transcription initiation, transcription termination, translational initiation, and operator sites related to expression of genes.

42. The method of claim 16, wherein the number of cycles is fewer than 100 cycles.

43. A method for shuffling polynucleotides, comprising

(a) providing at least two variants of a polynucleotide as starting substrates to be shuffled;

and

(b) shuffling the at least two variant polynucleotides by contacting the variants with a population of completely or partially random primers, and conducting a multi-cyclic polynucleotide extension reaction wherein

(i) in at least one cycle, the primers anneal to the variant forms and primer replication of the variants thereby generating extended primers, wherein the at least one cycle is terminated by an increase in temperature to terminate extension; and

(ii) in at least one subsequent cycle, the extended primers generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations forming annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form further extended primers;

whereby the polynucleotide extension is continued for sufficient cycles until the further extended primers includes recombinant forms of the shuffled polynucleotide.

44. The method of claim 43, wherein the temperature is raised to between 90°

C. and 99°

C.

45. The method of claim 43, wherein the starting substrates are labeled, and the separated from the extended primers generated in step (b) by use of the label.

46. The method of claim 43, wherein the multi-cyclic polynucleotide extension reaction is conducted under conditions promoting misincorporation of nucleotides.

47. The method of claim 43, wherein the starting substrates have a length of 50 bp to 20 kb.

48. The method of claim 43, wherein the multi-cyclic polynucleotide extension reaction is conducted with a polymerase selected from the group consisting of T4 polymerase, T7 polymerase, E. coli DNA polymerase I and the Klenow fragment of DNA polymerase I, and wherein said polymerase is added to the reaction mixture after each cycle.

49. The method of claim 43, wherein the multi-cyclic polynucleotide extension reaction is conducted in the presence of an RNA polymerase.

50. The method of claim 43, wherein said polymerase is an error-prone polymerase.

51. The method of claim 43, wherein the starting substrates are natural variants.

52. The method of claim 43, wherein at least two starting substrates exhibit more than 50% sequence identity.

53. The method of claim 52, wherein at least two starting substrates exhibit more than 70% sequence identity.

54. The method of claim 53, wherein at least two starting substrates exhibit more than 90% sequence identity.

55. The method of claim 54, wherein at least two starting substrates exhibit more than 95% sequence identity.

56. The method of claim 43, wherein the primers are labeled.

57. The method of claim 56, wherein the primers are 10 to 70 bp.

58. The method of claim 56, wherein the label is biotin or digoxigenin.

59. The method of claim 58, wherein the primers are 15 to 40 bp.

60. The method of claim 43, wherein the primers are 6 to 200 bp.

61. The method of claim 43, wherein the substrates are from naturally occurring organisms of different species.

62. The method of claim 43, wherein the starting substrates are linear DNA fragments generated by a PCR reaction.

63. The method of claim 43, wherein the starting substrates are cloned into vectors.

64. The method of claim 43, wherein the starting substrates encode variant forms of an enzyme.

65. The method of claim 64, wherein enzyme is selected from the group consisting of carbonyl hydrolase, carbohydrase, an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.

66. The method of claim 43, wherein the starting substrates encode variant forms of a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

67. The method of claim 43, wherein the starting substrates have a function selected from the group consisting of transcription initiation, transcription termination, translational initiation, and operator sites related to expression of genes.

68. The method of claim 43, wherein the number of cycles is fewer than 100 cycles.

69. A method for shuffling polynucleotides, comprising:

(a) providing at least two variants of a polynucleotide to be shuffled; and

(b) shuffling the at least two variant polynucleotides by contacting the variants with a population of completely or partially random primers, and conducting a multi-cyclic polynucleotide extension reaction wherein

(i) in at least one cycle, the primers anneal to the variants and prime replication of the variants thereby generating extended primers, wherein at least one cycle is conducted with the Klenow fragment of DNA polymerase I; and

(ii) in at least one subsequent cycle, the extended primers generated in a previous cycle are denatured in single-stranded fragments which anneal in new combinations forming annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form further extended primers;

whereby the polynucleotide extension is continued for sufficient cycles until the further extended primers includes recombinant forms of the shuffled polynucleotide.

70. The method of claim 69, wherein the starting substrates are labeled, and the separated from the extended primers generated in step (b) by use of the label.

71. The method of claim 69, wherein the multi-cyclic polynucleotide extension reaction is conducted under conditions promoting misincorporation of nucleotides.

72. The method of claim 69, wherein the starting substrates have a length of 50 bp to 20 kb.

73. The method of claim 69, wherein the starting substrates are natural variants.

74. The method of claim 69, wherein at least two starting substrates exhibit more than 50% sequence identity.

75. The method of claim 74, wherein at least two starting substrates exhibit more than 70% sequence identity.

76. The method of claim 75, wherein at least two starting substrates exhibit more than 90% sequence identity.

77. The method of claim 76, wherein at least two starting substrates exhibit more than 95% sequence identity.

78. The method of claim 69, wherein the primers are labeled.

79. The method of claim 78, wherein the label is biotin or digoxigenin.

80. The method of claim 69, wherein the primers are 6 to 200 bp in length.

81. The method of claim 80, wherein said primers are 10 to 70 bp in length.

82. The method of claim 81, wherein said primers are 15 to 40 bp in length.

83. The method of claim 69, wherein the substrates are from naturally occurring organisms of different species.

84. The method of claim 69, wherein the starting substrates are linear DNA fragments generated by a PCR reaction.

85. The method of claim 69, wherein the starting substrates are cloned into vectors.

86. The method of claim 69, wherein the starting substrates encode variant forms of an enzyme.

87. The method of claim 86, wherein enzyme is selected from the group consisting of carbonyl hydrolase, carbohydrase, an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.

88. The method of claim 69, wherein the starting substrates encode variant forms of a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

89. The method of claim 69, wherein the starting substrates have a function selected from the group consisting of transcription initiation, transcription termination, translational initiation, and operator sites related to expression of genes.

90. The method of claim 69, wherein the number of cycles is fewer than 100 cycles.

91. A method of shuffling polynucleotides, comprising:

shuffling first strands of a population of polynucleotides without shuffling second strands; and

synthesizing strands complementary to the shuffled first strands to form shuffled duplex polynucleotides.

92. The method of claim 91, wherein the first strands are isolated from the second strands by labeling the with biotin.

93. The method of claim 91, wherein the first strands are a pool of polynucleotides comprising diverse forms of a polynucleotide.

94. The method of claim 93, wherein the diverse forms of the polynucleotide are from naturally occurring organisms of different species.

95. The method of claim 91, wherein the pool of polynucleotides exhibit more than 50% sequence identity.

96. The method of claim 95, wherein the pool of polynucleotides exhibit more than 70% sequence identity.

97. The method of claim 96, wherein the pool of polynucleotides exhibit more than 90% sequence identity.

98. The method of claim 97, wherein the pool of polynucleotides exhibit more than 95% sequence identity.

99. The method of claim 91, wherein the first strands are contacted with at least one completely random primer.

100. The method of claim 91, wherein the first strands are contacted with at least one partly random primer or at least a pair of partly random primers.

101. A method of identifying polypeptides exhibiting a desired property, comprising the method of claim 91, further comprising expressing and screening a polypeptide encoded by the shuffled polynucleotide for a desired property.

102. The method of claim 101, wherein the property is an enzymatic activity.

103. The method of claim 91, wherein at least one extension cycle is conducted under conditions of incomplete elongation.

104. The method of claim 91, wherein the population of polynucleotides encode variant forms of an enzyme.

105. The method of claim 104, wherein enzyme is selected from the group consisting of carbonyl hydrolase, carbohydrase, an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.

106. The method of claim 105, wherein the population of polynucleotides comprises at least two variant polynucleotides and wherein the variant polynucleotides encode a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

107. In a method of shuffling polynucleotides from variants by template directed polynucleotide extension of a population of fragments of polynucleotides, the improvement wherein opposing strands of polynucleotides to be shuffled are separated before conducting the shuffling step, and the shuffling reaction is conducted in the presence of only one strand from each variant.

108. The method of claim 107, wherein the opposing strands are separated by labeling with biotin.

109. The method of claim 107, wherein the polynucleotides to be shuffled exhibit more than 50% sequence identity.

110. The method of claim 109, wherein the polynucleotides to be shuffled exhibit more than 70% sequence identity.

111. The method of claim 110, wherein the polynucleotides to be shuffled exhibit more than 90% sequence identity.

112. The method of claim 111, wherein the polynucleotides to be shuffled exhibit more than 95% sequence identity.

113. The method of claim 107, wherein the strands to be shuffled are contacted with at least one completely random primer.

114. The method of claim 107, wherein the strands to be shuffled are contacted with at least one partly random primer or at least a pair of partly random primers.

115. The method of claim 107, wherein the strands to be shuffled are contacted with at least one primer 6 to 200 bp in length.

116. The method of claim 115, wherein the strands to be shuffled are contacted with at least one primer 10 to 70 bp in length.

117. The method of claim 116, wherein the strands to be shuffled are contacted with at least one primer 15 to 40 bp in length.

118. A method of identifying polypeptides exhibiting a desired property, comprising the method of claim 107, further comprising expressing and screening a polypeptide encoded by the shuffled polynucleotide for a desired property.

119. The method of claim 118, wherein the property is an enzymatic activity.

120. The method of claim 107, wherein the variant polynucleotides are from naturally occurring organisms of different species.

121. The method of claim 107, wherein the shuffling reaction is performed With at least one extension cycle conducted under conditions of incomplete elongation.

122. The method of claim 107, wherein the population of polynucleotides encode diverse forms of an enzyme.

123. The method of claim 122, wherein enzyme is selected from the group consisting of carbonyl hydrolase, carbohydrase, an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.

124. The method of claim 107, wherein the population of polynucleotides comprises at least two variant polynucleotides and wherein the variant polynucleotides encode a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pituary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO) and prolactin.

125. A method for shuffling polynucleotides, comprising:

(a) isolating a sense strand from an at least one first polynucleotide variant and an antisense strand from an at least one second polynucleotide variant, wherein only the isolated strands are templates for shuffling;

(b) shuffling the isolated strands of the polynucleotide variants by contacting the variant polynucleotides with a population of primers and conducting a multi-cyclic polynucleotide extension to form shuffled double-stranded polynucleotides, wherein at least one cycle is conducted with a shorter extension time than required for complete extension of the templates.

126. A method for shuffling polynucleotides, comprising:

(a) isolating a sense strand from an at least one first polynucleotide variant and an antisense strand from at least one second polynucleotide variant, wherein only the isolated strands are templates for shuffling;

(b) shuffling the isolated strands of the polynucleotide variants by contacting the variants with a population of primers and conducting a multi-cyclic polynucleotide extension to form shuffled double-stranded polynucleotides, wherein at least one cycle is terminated by an increase in temperature to terminate extension.