Enzymatic synthesis of oligonucleotides using digestible templates

• US 5,932,450 A
• Filed: 06/07/1995
• Issued: 08/03/1999
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Kacian, Daniel L., Dattagupta, Nanibhushan, Lackey, David Bruce
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/484,519
• Time in Patent Office: 1,518 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 514/44, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

The present invention describes methods of synthesizing oligonucleotides. In particular, it relates to methods of enzymatically synthesizing chirally pure oligonucleotides using templates which can be digested after synthesis. By using digestible templates, separation and purification of the synthesized oligonucleotides are greatly facilitated.

Claims

1. A method of synthesizing two or more oligonucleotides comprising the steps of:

(a) providing a nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' to said primer binding region, said primer binding region being complementary to a nucleic acid primer and said oligonucleotide complementary region comprising two or more subregions, each said subregion being complementary to an oligonucleotide to be synthesized, wherein said template is used to direct the synthesis of a primer extension product comprising;

an oligonucleotide product comprising said oligonucleotides and one or more cleavage sites, each said cleavage site separating a pair of said oligonucleotides positioned adjacent to one another on said oligonucleotide product;

a nucleic acid primer; and

a cleavage site separating said primer from said oligonucleotide product;

(b) contacting said template with said primer under conditions such that said primer is able to hybridize to the primer binding region of said template to form a template-primer hybrid;

(c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form said primer extension product;

(d) cleaving said primer extension product such that said primer is separated from said oligonucleotide product and the oligonucleotides of said oligonucleotide product are separated from each other; and

(e) digesting said template before, simultaneous with or subsequent to step (d), so that said template is separated from said primer extension product.

2. The method of claim 1, wherein said oligonucleotides are modified.

3. The method of claim 2, wherein said oligonucleotides are phosphorothioate-containing oligonucleotides.

4. The method of claim 2, wherein said oligonucleotides are phosphorothioate oligonucleotides.

5. The method of claim 4, wherein said oligonucleotides are chirally pure.

6. The method of claim 1, wherein said template is RNA.

7. The method of claim 6, wherein said template is digested using an RNAse H activity or alkaline hydrolysis.

8. The method of claim 2, 3 or 4, wherein said cleavage site separating said primer from said oligonucleotide product comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

9. The method of claim 2, 3 or 4, wherein at least one of said cleavage sites separating said adjacent oligonucleotides comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

10. The method of claim 1, wherein said primer is DNA and at least one ribonucleotide located at or near the 3'"'"'-terminus of said primer.

11. The method of claim 10, wherein said primer is cleaved from said oligonucleotide product using an RNAse H activity or alkaline hydrolysis.

12. The method of claim 1, wherein said primer contains at least one deoxyuridine at or near its 3'"'"'-terminus.

13. The method of claim 12, wherein said primer is cleaved from said oligonucleotide product using a DNA glycosylase and an AP-endonuclease.

14. The method of claim 1 or 6, wherein said template and said primer are contained on the same nucleic acid strand, such that said template is a self-priming template comprising a self-complementary region and an oligonucleotide complementary region, and wherein said primer comprises the 3'"'"'-terminus of said self-complementary region.

15. The method of claim 1, wherein said primer extension product is cleaved at one or more cleavage sites using alkaline hydrolysis.

16. A method of synthesizing an oligonucleotide comprising the steps of:

(a) providing a nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' to said primer binding region, said primer binding region being complementary to a nucleic acid primer and said oligonucleotide complementary region being complementary to an oligonucleotide to be synthesized,

wherein said primer contains a deoxyuridine located at or near the 3'"'"'-terminus of said primer, and

wherein said template is used to direct the synthesis of a primer extension product comprising said oligonucleotide, said primer, and a cleavage site separating said primer from said oligonucleotide;

(b) contacting said template with said primer under conditions such that said primer is able to hybridize to the primer binding region of said template to form a template-primer hybrid;

(c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form a primer extension product;

(d) cleaving said primer extension product at said cleavage site to separate said primer from said oligonucleotide; and

(e) digesting said template before, simultaneous with or subsequent to step (d), so that said template is separated from said primer extension product.

17. The method of claim 16, wherein said oligonucleotide is modified.

18. The method of claim 17, wherein said oligonucleotide is a phosphorothioate-containing oligonucleotide.

19. The method of claim 17, wherein said oligonucleotide is a phosphorothioate oligonucleotide.

20. The method of claim 19, wherein said oligonucleotide is chirally pure.

21. The method of claim 16, wherein said template is RNA.

22. The method of claim 21, wherein said template is digested using an RNAse H activity or alkaline hydrolysis.

23. The method of claim 17, 18 or 19, wherein said cleavage site separating said primer from said oligonucleotide product comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

24. The method of claim 16, wherein said primer is cleaved from said oligonucleotide product using a DNA glycosylase and an AP-endonuclease.

25. The method of claim 16 or 21, wherein said template and said primer are contained on the same nucleic acid strand, such that said template is a self-priming template comprising a self-complementary region and an oligonucleotide complementary region, and wherein said primer comprises the 3'"'"'-terminus of said self-complementary region.

26. The method of claim 16, wherein step (a) further includes providing a second nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' to said primer binding region, wherein the primer complementary region of said second template comprises the primer of said first template.

27. A method of synthesizing an oligonucleotide comprising the steps of:

(a) providing a nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' of said primer binding region, said primer binding region being complementary to a nucleic acid primer and said oligonucleotide complementary region being complementary to an oligonucleotide to be synthesized,

wherein said template and said primer are contained on the same nucleic acid strand, such that said template is a self-priming template comprising a self-complementary region and an oligonucleotide complementary region, said primer comprising the 3'"'"'-terminus of said self-complementary region, and

wherein said template is used to direct the synthesis of a primer extension product comprising said oligonucleotide, said primer, and a cleavage site separating said primer from said oligonucleotide;

(b) contacting said template with said primer under conditions such that said primer is able to hybridize to the primer binding region of said template to form a template-primer hybrid;

(c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form a primer extension product;

(d) cleaving said primer extension product at said cleavage site to separate said primer from said oligonucleotide; and

(e) digesting said template before, simultaneous with or subsequent to step (d), so that said template is separated from said primer extension product.

28. The method of claim 27, wherein said oligonucleotide is modified.

29. The method of claim 28, wherein said oligonucleotide is a phosphorothioate-containing oligonucleotide.

30. The method of claim 28, wherein said oligonucleotide is a phosphorothioate oligonucleotide.

31. The method of claim 30, wherein said oligonucleotide is chirally pure.

32. The method of claim 27, wherein said template is RNA.

33. The method of claim 32, wherein said template is digested using an RNAse H activity or alkaline hydrolysis.

34. The method of claim 28, 29 or 30, wherein said cleavage site separating said primer from said oligonucleotide product comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

35. The method of claim 27, wherein said primer comprises DNA and a ribonucleotide located at or near the 3'"'"'-terminus of said primer.

36. The method of claim 35, wherein said primer is separated from said oligonucleotide using alkaline hydrolysis or an RNAse H activity.

37. The method of claim 27, wherein said primer contains a deoxyuridine located at or near the 3'"'"'-terminus of said primer.

38. The method of claim 37, wherein said primer is cleaved from said oligonucleotide product using a DNA glycosylase and an AP-endonuclease.

39. A method of synthesizing an oligonucleotide comprising the steps of:

(a) providing a nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' of said primer binding region, said primer binding region being complementary to a nucleic acid primer and said oligonucleotide complementary region being complementary to an oligonucleotide to be synthesized,

wherein said primer comprises DNA and a ribonucleotide located at or near the 3'"'"'-terminus of said primer, and

wherein said template is used to direct the synthesis of a primer extension product comprising said oligonucleotide, said primer, and a cleavage site separating said primer from said oligonucleotide;

(b) contacting said template with said primer under conditions such that said primer is able to hybridize to the primer binding region of said template to form a template-primer hybrid;

(c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form a primer extension product;

(d) cleaving said primer extension product at said cleavage site to separate said primer from said oligonucleotide; and

(e) digesting said template before, simultaneous with or subsequent to step (d), so that said template is separated from said primer extension product.

40. The method of claim 39, wherein said oligonucleotide is modified.

41. The method of claim 40, wherein said oligonucleotide is a phosphorothioate-containing oligonucleotide.

42. The method of claim 40, wherein said oligonucleotide is a phosphorothioate oligonucleotide.

43. The method of claim 42, wherein said oligonucleotide is chirally pure.

44. The method of claim 39, wherein said template is RNA.

45. The method of claim 44, wherein said template is digested using an RNAse H activity or alkaline hydrolysis.

46. The method of claim 40, 41 or 42, wherein said cleavage site separating said primer from said oligonucleotide product comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

47. The method of claim 39, wherein said primer is separated from said oligonucleotide using alkaline hydrolysis or an RNAse H activity.

48. The method of claim 39 or 44, wherein said template and said primer are contained on the same nucleic acid strand, such that said template is a self-priming template comprising a self-complementary region and an oligonucleotide complementary region, and wherein said primer comprises the 3'"'"'-terminus of said self-complementary region.

49. The method of claim 39, wherein step (a) further includes providing a second nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' to said primer binding region, wherein the primer complementary region of said second template comprises the primer of said first template.

50. A method of synthesizing an oligonucleotide comprising the steps of:

(a) providing a nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' of said primer binding region, said primer binding region being complementary to a nucleic acid primer and said oligonucleotide complementary region being complementary to an oligonucleotide to be synthesized,

wherein said template is used to direct the synthesis of a primer extension product comprising said oligonucleotide, said primer, and a cleavage site separating said primer from said oligonucleotide,

wherein said cleavage site comprises a restriction endonuclease recognition sequence, and

wherein said primer is separated from said oligonucleotide using a restriction endonuclease able to recognize said restriction endonuclease recognition sequence;

(b) contacting said template with said primer under conditions such that said primer is able to hybridize to the primer binding region of said template to form a template-primer hybrid;

(c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form a primer extension product;

(d) cleaving said primer extension product at said cleavage site to separate said primer from said oligonucleotide; and

(e) digesting said template before, simultaneous with or subsequent to step (d), so that said template is separated from said primer extension product.

51. The method of claim 50, wherein said oligonucleotide is modified.

52. The method of claim 51, wherein said oligonucleotide is a phosphorothioate-containing oligonucleotide.

53. The method of claim 51, wherein said oligonucleotide is a phosphorothioate oligonucleotide.

54. The method of claim 53, wherein said oligonucleotide is chirally pure.

55. The method of claim 50, wherein said template is RNA.

56. The method of claim 55, wherein said template is digested using an RNAse H activity or alkaline hydrolysis.

57. The method of claim 50 or 55, wherein said template and said primer are contained on the same nucleic acid strand, such that said template is a self-priming template comprising a self-complementary region and an oligonucleotide complementary region, and wherein said primer comprises the 3'"'"'-terminus of said self-complementary region.

58. The method of claim 50, wherein step (a) further includes providing a second nucleic acid template comprising a primer binding region and an oligonucleotide complementary region located 5'"'"' to said primer binding region, wherein the primer complementary region of said second template comprises the primer of said first template.

59. A method of synthesizing at least one oligonucleotide comprising the steps of:

(a) providing first and second nucleic acid templates, each said template comprising a primer complementary region and an oligonucleotide complementary region located 5'"'"' to the primer complementary region, under conditions such that the primer complementary region of said first template is able to hybridize with the primer complementary region of said second template, thus forming a hybrid between the primer complementary regions of said first and second templates,

wherein the primer complementary region of said first template comprises a primer for said second template,

wherein the primer complementary region of said second template comprises a primer for said first template,

wherein each said oligonucleotide complementary region is complementary to an oligonucleotide to be synthesized, said oligonucleotides being the same or different, and

wherein each said template is used to direct the synthesis of a primer extension product comprising said oligonucleotide, said primer, and a cleavage site separating said primer from said oligonucleotide;

(b) incubating said hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form said primer extension products;

(c) cleaving said primer extension products such that said primers are separated from said oligonucleotides; and

(d) digesting said templates before, simultaneous with or subsequent to step (c), so that said templates are separated from said primer extension products.

60. The method of claim 59, wherein said oligonucleotides are modified.

61. The method of claim 60, wherein said oligonucleotides are phosphorothioate-containing oligonucleotides.

62. The method of claim 60, wherein said oligonucleotides are phosphorothioate oligonucleotides.

63. The method of claim 62, wherein said oligonucleotides are chirally pure.

64. The method of claim 59, wherein said templates are RNA.

65. The method of claim 63, wherein said templates are digested using an RNAse H activity or alkaline hydrolysis.

66. The method of claim 60, 61 or 62, wherein said cleavage sites separating said primers from said oligonucleotides comprise restriction endonuclease recognition sequences, each said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

67. The method of claim 59, wherein said primers are DNA and at least one ribonucleotide located at or near the 3'"'"'-terminus of said primers.

68. The method of claim 67, wherein said primers are cleaved from said oligonucleotides using an RNAse H activity or alkaline hydrolysis.

69. The method of claim 59, wherein said primers contain at least one deoxyuridine at or near its 3'"'"'-terminus.

70. The method of claim 69, wherein said primers are cleaved from said oligonucleotides using a DNA glycosylase and an AP-endonuclease.

71. A method of synthesizing two or more oligonucleotides comprising the steps of:

(a) providing first and second nucleic acid templates, each said template comprising a primer complementary region and an oligonucleotide complementary region located 5'"'"' to the primer complementary region, under conditions such that the primer complementary region of said first template is able to hybridize with the primer complementary region of said second template, thus forming a hybrid between the primer complementary regions of said first and second templates,

wherein the primer complementary region of said first template comprises a primer for said second template,

wherein the primer complementary region of said second template comprises a primer for said first template,

wherein each said oligonucleotide complementary region comprises two or more subregions, each said subregion being complementary to an oligonucleotide, and

wherein each said template is used to direct the synthesis of a primer extension product comprising;

an oligonucleotide product comprising two or more of said oligonucleotides and one or more cleavage sites, each said cleavage site separating a pair of said oligonucleotides positioned adjacent to one another on said oligonucleotide product;

said primer; and

a cleavage site separating said primer from said oligonucleotide product;

(b) incubating said hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form said primer extension products;

(c) cleaving said primer extension products such that said primers are separated from said oligonucleotide products and the oligonucleotides of said oligonucleotide products are separated from each other; and

(d) digesting said templates before, simultaneous with or subsequent to step (c), so that said templates are separated from said primer extension products.

72. The method of claim 71, wherein said oligonucleotides are modified.

73. The method of claim 72, wherein said oligonucleotides are phosphorothioate-containing oligonucleotides.

74. The method of claim 72, wherein said oligonucleotides are phosphorothioate oligonucleotides.

75. The method of claim 71, wherein said templates are RNA.

76. The method of claim 75, wherein said templates are digested using an RNAse H activity or alkaline hydrolysis.

77. The method of claim 71, 72, 73, 74, 75 or 76, wherein said oligonucleotides are chirally pure.

78. The method of claim 72, 73 or 74, wherein said templates are RNA.

79. The method of claim 78, wherein said oligonucleotides are chirally pure.

80. The method of claim 72, 73 or 74, wherein said cleavage sites separating said primers from said oligonucleotide products comprise restriction endonuclease recognition sequences, each said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

81. The method of claim 72, 73 or 74, wherein at least one of said cleavage sites separating said adjacent oligonucleotides comprises a restriction endonuclease recognition sequence, said recognition sequence being cleavable with a restriction endonuclease able to recognize said recognition sequence.

82. The method of claim 71, wherein said primers are cleaved from said oligonucleotide products using an RNAse H activity or alkaline hydrolysis.

83. The method of claim 71, wherein said primers are DNA and at least one ribonucleotide located at or near the 3'"'"'-terminus of said primers.

84. The method of claim 71, wherein said primers contain at least one deoxyuridine at or near its 3'"'"'-terminus.

85. The method of claim 71, wherein said primer are cleaved from said oligonucleotide products using a DNA glycosylase and an AP-endonuclease.

86. The method of claim 71, wherein said primer extension products are cleaved at one or more cleavage sites using alkaline hydrolysis.